CN106459010B - Quinoxaline derivatives as FGFR kinase modulators - Google Patents

Abstract

The present invention relates to novel quinoxaline derivative compounds of formula (I), pharmaceutical compositions comprising said compounds, processes for preparing said compounds and the use of said compounds in the treatment of diseases, such as cancer.

Description

Quinoxaline derivatives as FGFR kinase modulators

Technical Field

The present invention relates to novel quinoxaline derivative compounds, pharmaceutical compositions comprising said compounds, processes for preparing said compounds and the use of said compounds in the treatment of diseases, such as cancer.

Disclosure of Invention

According to a first aspect of the invention, there is provided a compound of formula (I):

(I)

including any tautomeric or stereochemically isomeric form thereof, wherein

n represents an integer equal to 1 or 2;

R₁represents hydrogen, C_1-6Alkyl, hydroxy C_1-6Alkyl, with-C (= O) NHCH₃Substituted C_1-6Alkyl or by-S (= O)₂-C_1-4Alkyl substituted C_1-6An alkyl group;

R_2arepresents hydrogen, fluorine or chlorine;

R_2bor R_2cEach independently represents a methoxy group or a hydroxyl group;

R₃represents hydrogen, C_1-6Alkyl radical, C_3-6Cycloalkyl radicals or with C_3-6Cycloalkyl-substituted C_1-2An alkyl group;

R₄represents hydrogen, methyl or ethyl;

a pharmaceutically acceptable salt thereof or a solvate thereof.

In one embodiment, compounds of formula (Ia) are provided:

(Ia)

including any tautomeric or stereochemically isomeric form thereof, wherein

n represents an integer equal to 1 or 2;

R₁represents hydrogen, C_1-6Alkyl, hydroxy C_1-6Alkyl, with-C (= O) NHCH₃Substituted C_1-6Alkyl or by-S (= O)₂-C_1-4Alkyl substituted C_1-6An alkyl group;

R_2arepresents hydrogen, fluorine or chlorine;

R_2bor R_2cEach independently represents a methoxy group or a hydroxyl group;

R₃represents hydrogen, C_1-6Alkyl radical, C_3-6Cycloalkyl radicals or with C_3-6Cycloalkyl-substituted C_1-2An alkyl group;

a pharmaceutically acceptable salt thereof or a solvate thereof.

In one embodiment, compounds of formula (Ib) are provided:

(Ib)

including any tautomeric or stereochemically isomeric form thereof, wherein

n represents an integer equal to 1 or 2;

R₁represents hydrogen, C_1-6Alkyl, hydroxy C_1-6Alkyl, with-C (= O) NHCH₃Substituted C_1-6Alkyl or by-S (= O)₂-C_1-4Alkyl substituted C_1-6An alkyl group;

R_2bor R_2cEach independently represents a methoxy group or a hydroxyl group;

R₃represents hydrogen, C_1-6Alkyl radical, C_3-6Cycloalkyl radicals or with C_3-6Cycloalkyl-substituted C_1-2An alkyl group;

a pharmaceutically acceptable salt thereof or a solvate thereof.

In one embodiment, compounds of formula (Ic) are provided:

(Ic)

including any tautomeric or stereochemically isomeric form thereof, wherein

R₁Represents hydrogen, C_1-6Alkyl, hydroxy C_1-6Alkyl, with-C (= O) NHCH₃Substituted C_1-6Alkyl or by-S (= O)₂-C_1-4Alkyl substituted C_1-6An alkyl group;

R_2bor R_2cEach independently represents a methoxy group or a hydroxyl group;

a pharmaceutically acceptable salt thereof or a solvate thereof.

In one embodiment, compounds of formula (Id) are provided:

(Id)

including any tautomeric or stereochemically isomeric form thereof, wherein

R_2bOr R_2cEach independently represents a methoxy group or a hydroxyl group;

a pharmaceutically acceptable salt thereof or a solvate thereof.

In one embodiment, there is provided a compound of formula (Ie):

(Ie)

including any tautomeric or stereochemically isomeric form thereof, wherein

R₁Represents hydrogen, C_1-6Alkyl, hydroxy C_1-6Alkyl, with-C (= O) NHCH₃Substituted C_1-6Alkyl or by-S (= O)₂-C_1-4Alkyl substituted C_1-6An alkyl group;

R_2bor R_2cEach independently represents a methoxy group or a hydroxyl group;

a pharmaceutically acceptable salt thereof or a solvate thereof.

WO2006/092430, WO2008/003702, WO01/68047, WO2005/007099, WO2004/098494, WO2009/141386, WO2004/030635, WO2008/141065, WO2011/026579, WO2011/028947, WO2007/003419, WO00/42026, WO2012/154760, WO2011/047129, WO2003/076416, WO2002/096873, WO2000/055153, EP548934, US4166117, WO2011/135376, WO2012/073017, WO2013/061074, WO2013/061081, WO2013/061077, WO2013/061080, WO2013/179034, WO2013/179033, WO2014/174307, each of which discloses a series of heterocyclyl derivatives.

Detailed Description

Unless the context indicates otherwise, reference to formula (I) in all parts of this document, including uses, methods and other aspects of the invention, includes reference to all other sub-formulae (e.g. Ia, Ib, Ic, Id), sub-formulae, preferences, embodiments and examples defined herein.

The prefix "C" as used herein_x-y"(wherein x and y are integers) refers to the number of carbon atoms in a given group. Thus, C_1-6Alkyl containing 1 to 6 carbon atoms, C_3-6Cycloalkyl radicals containing from 3 to 6 carbon atomsSub, hydroxy C_1-6Alkyl groups contain 1-6 carbon atoms, and the like.

The term "C" as used herein as a group or part of a group_1-2Alkyl group "," C_1-4Alkyl "or" C_1-6Alkyl "refers to a linear or branched saturated hydrocarbon group containing 1 or 2, or 1 to 4, or 1 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or hexyl and the like.

The term "C" as used herein_3-6Cycloalkyl "means a saturated monocyclic hydrocarbon ring of 3 to 6 carbon atoms. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

The term "hydroxy C" as used herein as a group or part of a group_1-4Alkyl "or" hydroxy C_1-6Alkyl "means C as defined herein_1-4Alkyl or C_1-6Alkyl in which one or more than one hydrogen atom is replaced by a hydroxyl group. Thus the term "hydroxy C_1-4Alkyl "or" hydroxy C_1-6Alkyl "includes monohydroxy C_1-4Alkyl, monohydroxy C_1-6Alkyl and polyhydroxy C_1-4Alkyl and polyhydroxy C_1-6An alkyl group. 1.2, 3 or more hydrogen atoms may be replaced by hydroxyl groups, thus hydroxyl groups C_1-4Alkyl or hydroxy C_1-6The alkyl group may have 1,2, 3, or more hydroxyl groups. Examples of such groups include hydroxymethyl, hydroxyethyl, hydroxypropyl and the like.

In one embodiment, in the compound of formula (I), n represents an integer equal to 1.

In one embodiment, in the compound of formula (I), n represents an integer equal to 2.

In one embodiment, in the compounds of formula (I), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly methyl.

In one embodiment, in the compounds of formula (I), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly ethyl。

In one embodiment, in the compounds of formula (I), R₁Represents hydrogen.

In one embodiment, in the compounds of formula (I), R_2aRepresents hydrogen or fluorine.

In one embodiment, in the compounds of formula (I), R_2aRepresents hydrogen.

In one embodiment, in the compounds of formula (I), R_2aRepresents fluorine.

In one embodiment, in the compounds of formula (I), R_2bRepresents a methoxy group.

In one embodiment, in the compounds of formula (I), R_2bRepresents a hydroxyl group.

In one embodiment, in the compounds of formula (I), R_2cRepresents a methoxy group.

In one embodiment, in the compounds of formula (I), R_2cRepresents a hydroxyl group.

In one embodiment, in the compounds of formula (I), R_2bRepresents methoxy and R_2cRepresents a hydroxyl group.

In one embodiment, in the compounds of formula (I), R_2bRepresents a hydroxyl group and R_2cRepresents a methoxy group.

In one embodiment, in the compounds of formula (I), R_2bAnd R_2cBoth represent methoxy.

In one embodiment, in the compounds of formula (I), R_2bAnd R_2cAll represent hydroxyl groups.

In one embodiment, in the compounds of formula (I), R₃Represents hydrogen.

In one embodiment, in the compounds of formula (I), R₃Is represented by C_1-6Alkyl, especially C_1-4Alkyl, even more particularly isopropyl.

In one embodiment, in the compounds of formula (I), R₃Is represented by C_1-6Alkyl, especially C_1-4Alkyl, even more particularly methyl.

In one embodiment, in the compounds of formula (I), R₄Represents hydrogen.

In one embodiment, in the compounds of formula (I), R₄Represents a methyl group or an ethyl group.

In one embodiment, in the compounds of formula (I),

n represents an integer equal to 1;

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl;

R_2arepresents hydrogen or fluorine, in particular hydrogen;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group;

R₃represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly C_1-4Alkyl, even more particularly isopropyl;

R₄represents hydrogen.

In one embodiment, in the compounds of formula (I),

n represents an integer equal to 1 or 2;

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl or ethyl;

R_2arepresents hydrogen or fluorine, in particular hydrogen;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group;

R₃represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly C_1-4Alkyl, even more particularly isopropyl or methyl;

R₄represents hydrogen.

In one embodiment, in the compound of formula (Ia), n represents an integer equal to 1.

In one embodiment, in the compound of formula (Ia), n represents an integer equal to 2.

In one embodimentIn the compound of the formula (Ia), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly methyl.

In one embodiment, in the compound of formula (Ia), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly ethyl.

In one embodiment, in the compound of formula (Ia), R₁Represents hydrogen.

In one embodiment, in the compound of formula (Ia), R_2aRepresents hydrogen or fluorine.

In one embodiment, in the compound of formula (Ia), R_2aRepresents hydrogen.

In one embodiment, in the compound of formula (Ia), R_2aRepresents fluorine.

In one embodiment, in the compound of formula (Ia), R_2bRepresents a methoxy group.

In one embodiment, in the compound of formula (Ia), R_2bRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ia), R_2cRepresents a methoxy group.

In one embodiment, in the compound of formula (Ia), R_2cRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ia), R_2bRepresents methoxy and R_2cRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ia), R_2bRepresents a hydroxyl group and R_2cRepresents a methoxy group.

In one embodiment, in the compound of formula (Ia), R_2bAnd R_2cBoth represent methoxy.

In one embodiment, in the compound of formula (Ia), R_2bAnd R_2cAll represent hydroxyl groups.

In one embodiment, in the compound of formula (Ia), R₃Represents hydrogen.

In one embodimentIn the compound of the formula (Ia), R₃Is represented by C_1-6Alkyl, especially C_1-4Alkyl, even more particularly isopropyl.

In one embodiment, in the compound of formula (Ia), R₃Is represented by C_1-6Alkyl, especially C_1-4Alkyl, even more particularly methyl.

In one embodiment, in the compound of formula (Ia),

n represents an integer equal to 1;

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl;

R_2arepresents hydrogen or fluorine, in particular hydrogen;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group;

R₃represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly C_1-4Alkyl, even more particularly isopropyl.

In one embodiment, in the compound of formula (Ia),

n represents an integer equal to 1 or 2;

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl or ethyl;

R_2arepresents hydrogen or fluorine, in particular hydrogen;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group;

R₃represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly C_1-4Alkyl, even more particularly isopropyl or methyl.

In one embodiment, in the compound of formula (Ib), n represents an integer equal to 1.

In one embodiment, in the compound of formula (Ib), n represents an integer equal to 2.

In one embodiment, the compound of formula (Ib)In, R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly methyl.

In one embodiment, in the compounds of formula (Ib), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly ethyl.

In one embodiment, in the compounds of formula (Ib), R₁Represents hydrogen.

In one embodiment, in the compounds of formula (Ib), R_2bRepresents a methoxy group.

In one embodiment, in the compounds of formula (Ib), R_2bRepresents a hydroxyl group.

In one embodiment, in the compounds of formula (Ib), R_2cRepresents a methoxy group.

In one embodiment, in the compounds of formula (Ib), R_2cRepresents a hydroxyl group.

In one embodiment, in the compounds of formula (Ib), R_2bRepresents methoxy and R_2cRepresents a hydroxyl group.

In one embodiment, in the compounds of formula (Ib), R_2bRepresents a hydroxyl group and R_2cRepresents a methoxy group.

In one embodiment, in the compounds of formula (Ib), R_2bAnd R_2cBoth represent methoxy.

In one embodiment, in the compounds of formula (Ib), R_2bAnd R_2cAll represent hydroxyl groups.

In one embodiment, in the compounds of formula (Ib), R₃Represents hydrogen.

In one embodiment, in the compounds of formula (Ib), R₃Is represented by C_1-6Alkyl, especially C_1-4Alkyl, even more particularly isopropyl.

In one embodiment, in the compounds of formula (Ib), R₃Is represented by C_1-6Alkyl, especially C_1-4Alkyl, even more particularly methyl.

In one embodiment, in the compounds of formula (Ib),

n represents an integer equal to 1;

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group;

R₃represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly C_1-4Alkyl, even more particularly isopropyl.

In one embodiment, in the compounds of formula (Ib),

n represents an integer equal to 1 or 2;

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl or ethyl;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group;

R₃represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly C_1-4Alkyl, even more particularly isopropyl or methyl.

In one embodiment, in the compound of formula (Ic), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly methyl.

In one embodiment, in the compound of formula (Ic), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly ethyl.

In one embodiment, in the compound of formula (Ic), R₁Represents hydrogen.

In one embodiment, in the compound of formula (Ic), R_2bRepresents a methoxy group.

In one embodiment, in the compound of formula (Ic), R_2bRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ic), R_2cRepresents a methoxy group。

In one embodiment, in the compound of formula (Ic), R_2cRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ic), R_2bRepresents methoxy and R_2cRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ic), R_2bRepresents a hydroxyl group and R_2cRepresents a methoxy group.

In one embodiment, in the compound of formula (Ic), R_2bAnd R_2cBoth represent methoxy.

In one embodiment, in the compound of formula (Ic), R_2bAnd R_2cAll represent hydroxyl groups.

In one embodiment, in the compound of formula (Ic),

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group.

In one embodiment, in the compound of formula (Ic),

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl or ethyl;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group.

In one embodiment, in the compound of formula (Id), R_2bRepresents a methoxy group.

In one embodiment, in the compound of formula (Id), R_2bRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Id), R_2cRepresents a methoxy group.

In one embodiment, in the compound of formula (Id), R_2cRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Id), R_2bRepresents methoxy andR_2crepresents a hydroxyl group.

In one embodiment, in the compound of formula (Id), R_2bRepresents a hydroxyl group and R_2cRepresents a methoxy group.

In one embodiment, in the compound of formula (Id), R_2bAnd R_2cBoth represent methoxy.

In one embodiment, in the compound of formula (Id), R_2bAnd R_2cAll represent hydroxyl groups.

In one embodiment, in the compound of formula (Ie), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly methyl.

In one embodiment, in the compound of formula (Ie), R₁Represents hydrogen or C_1-6Alkyl, especially C_1-6Alkyl, more particularly ethyl.

In one embodiment, in the compound of formula (Ie), R₁Represents hydrogen.

In one embodiment, in the compound of formula (Ie), R_2bRepresents a methoxy group.

In one embodiment, in the compound of formula (Ie), R_2bRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ie), R_2cRepresents a methoxy group.

In one embodiment, in the compound of formula (Ie), R_2cRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ie), R_2bRepresents methoxy and R_2cRepresents a hydroxyl group.

In one embodiment, in the compound of formula (Ie), R_2bRepresents a hydroxyl group and R_2cRepresents a methoxy group.

In one embodiment, in the compound of formula (Ie), R_2bAnd R_2cBoth represent methoxy.

In one embodiment, in the compound of formula (Ie), R_2bAnd R_2cAll represent hydroxyl groups.

In one embodiment, in the compound of formula (Ie),

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group.

In one embodiment, in the compound of formula (Ie),

R₁is represented by C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl or ethyl;

R_2brepresents a methoxy group;

R_2crepresents a methoxy group.

In one embodiment, in the compounds of formula (I),

n represents an integer equal to 1 or 2;

R₁represents hydrogen or C_1-6Alkyl, especially C_1-4Alkyl, more particularly methyl or ethyl;

R_2arepresents hydrogen or fluorine, in particular hydrogen;

R_2brepresents methoxy or hydroxy;

R_2crepresents methoxy or hydroxy;

R₃is represented by C_1-6Alkyl, more particularly C_1-4Alkyl, even more particularly isopropyl or methyl;

R₄represents hydrogen.

In one embodiment, the compound of formula (I) as defined herein is selected from or is one of the following compounds:

a pharmaceutically acceptable salt thereof or a solvate thereof.

In one embodiment, the compound of formula (I) as defined herein is selected from or is one of the following compounds:

a pharmaceutically acceptable salt thereof or a solvate thereof.

For the avoidance of doubt, it is to be understood that each of the general and specific preferences, embodiments and examples for one substituent may be combined with each of the general and specific preferences, embodiments and examples for one or more, preferably all, of the other substituents defined herein, and all such embodiments are included in the present application.

Process for preparing compounds of formula (I)

In this section, as in all other sections of this application, reference to formula (I) also includes all other subclasses defined herein and embodiments thereof, unless the context indicates otherwise.

In general, the compounds of formula (I) may be prepared according to the following reaction scheme.

Scheme 1

In scheme 1, the following reaction conditions apply:

1: the intermediate of formula (II) is reacted with formaldehyde in a suitable solvent such as dioxane,N,N-dimethylformamide,N,N-reaction in the presence of dimethylacetamide at a temperature ranging from room temperature to reflux.

It is believed to be within the knowledge of the skilled person that it may be appropriate to identify the protecting group under what conditions and on what part of the molecule. For example, R₁Protecting groups on substituents or on pyrazole moieties, or R₃On a substituent or R_2a,b,cA protecting group on a substituent, or a combination thereof. It is also believed that the technician can identify the bestPossible protecting groups, e.g. -C (= O) -O-C_1-4Alkyl or

Or O-Si (CH)₃)₂(C(CH₃)₃) or-CH₂-O-CH₂CH₂-O-CH₃。

The invention also encompasses deuterated compounds. During synthesis, these deuterated compounds can be prepared by using suitable deuterated intermediates.

The compounds of formula (I) may also be converted to each other by reactions or functional group transformations known in the art.

Wherein R is₁A compound of formula (I) representing hydrogen by reaction with C in the presence of a suitable base such as sodium hydride or potassium carbonate and a suitable solvent such as acetonitrile or N, N-dimethylformamide_1-6alkyl-W or hydroxy-C_1-6alkyl-W reaction, convertible to wherein R₁Is represented by C_1-6Alkyl or hydroxy C_1-6Alkyl, wherein W represents a suitable leaving group, such as halogen, e.g. bromine and the like.

Wherein R is₁A compound of formula (I) representing hydrogen, by reaction with W-C in the presence of a suitable base such as sodium hydride and a suitable solvent such as N, N-dimethylformamide_1-6alkyl-O-Si (CH)₃)₂(C(CH₃)₃) Reactions followed by deprotection of the silyl protecting group by methods known in the art may also be converted to where R₁Is represented by C_1-6alkyl-OH compounds of formula (I).

Wherein R is₁A compound of formula (I) representing hydrogen, by reaction with C in the presence of a suitable base such as triethylamine and a suitable solvent such as an alcohol, e.g. methanol_1-6By reaction with alkyl-vinyl sulfones, or by reaction with C_1-6Alkyl-2-bromoethylsulfone can also be converted to a compound wherein R is in the presence of a suitable deprotonating agent such as NaH and a suitable solvent such as dimethylformamide₁Is represented by-S (= O)₂-C_1-6Alkyl substituted ethyl compounds of formula (I).

Wherein R is_2bOr R_2crepresents-OCH₃The compound of formula (I) can be converted to R wherein R is_2bOr R_2cA compound of formula (I) representing-OH.

Wherein R is_2bOr R_2cA compound of formula (I) representing-OH by reaction between a suitable base such as potassium carbonate and a suitable solvent such asN,N-dimethylformamide with methyl iodide in the presence of methyl iodide, convertible to R_2bOr R_2crepresents-OCH₃A compound of formula (I).

In general, the compounds of formula (Id) can also be prepared according to the following reaction scheme by incubating them with a liver portion of an animal, such as rat or human, and then isolating the desired product from the incubation medium.

Scheme 2

Intermediates of formula (II) or (II-a) may be prepared as described in WO2011/135376 (e.g. compounds of formula (I-b) or (I-b-3) of WO 2011/135376).

Another aspect of the present invention is a process for the preparation of a compound of formula (I) as defined herein, said process comprising:

(i) reacting a compound of formula (II) with formaldehyde in the presence of a suitable solvent such as dioxane, N-dimethylformamide, N-dimethylacetamide at a suitable temperature, for example a temperature in the range of room temperature to reflux;

wherein R is₁、R_2a、R_2b、R_2c、R₃、R₄And n is as defined herein; and optionally, thereafter converting one compound of formula (I) to another compound of formula (I).

Pharmaceutically acceptable salts, solvates or derivatives thereof

In this section, as in all other sections of this application, reference to formula (I) includes reference to all other subclasses, preferences, embodiments and examples thereof defined herein, unless otherwise indicated herein.

Unless otherwise indicated, reference to a particular compound also includes, for example, the ionic forms, salts, solvates, isomers, tautomers, esters, prodrugs, isotopes and protected forms thereof discussed below; preferably an ionic form or salt or tautomer or isomer or solvate thereof; more preferably its ionic form or salt or tautomer or solvate or protected form, even more preferably its salt or tautomer or solvate. Many of the compounds of formula (I) may exist in the form of salts, for example acid addition salts, or in some cases salts of organic and inorganic bases such as carboxylates, sulfonates and phosphates. All such salts are within the scope of the present invention, and reference to the compound of formula (I) includes salt forms of the compound. It will be appreciated that reference to "derivatives" includes reference to ionic forms, salts, solvates, isomers, tautomers, esters, prodrugs, isotopes and protected forms thereof.

One aspect of the present invention provides a compound as defined herein or a salt, tautomer or solvate thereof. Another aspect of the invention provides a compound as defined herein or a salt or solvate thereof. Reference to a compound of formula (I) and subclasses thereof as defined herein includes salts or solvates or tautomers of said compound.

Salt forms of the compounds of the invention are typically Pharmaceutically Acceptable Salts, Berge et al, 1977, "pharmaceutical Acceptable Salts",J. Pharm. Sciexamples of pharmaceutically acceptable salts are discussed at volume 66, pages 1-19. However, non-pharmaceutically acceptable salts may also be prepared in intermediate form, which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts which may be used, for example, in the purification or isolation of the compounds of the invention also form part of the invention.

Can be prepared by conventional chemical methods, e.g. by the methods described inPharmaceutical Salts: Properties, Selection, and UseThe salts of the invention are synthesized from parent compounds which contain basic or acidic moieties by the method of P.Heinrich Stahl (ed.), Camile G.Wermuth (ed.), ISBN: 3-90639-026-8, Hardcover, page 388, August 2002. The salts may generally be prepared by reacting the free acid or base forms of these compounds with a suitable base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile are used. The compounds of the invention may be present as mono-or di-salts depending on the pKa of the acid from which the salt is formed.

Acid addition salts can be formed with a wide variety of acids, both acid-free and organic. Examples of acid addition salts include salts with acids selected from the group consisting of: acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphoric acid, camphorsulfonic acid, (+) - (1S) -camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), alpha-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, isethionic acid, D-gluconic acid, Lactic acid (e.g., (+) -L-lactic acid and (. + -.) -DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-) -L-malic acid, malonic acid, (+ -.) -DL-mandelic acid, methanesulfonic acid, naphthalenesulfonic acid (e.g., naphthalene-2-sulfonic acid), naphthalene-1, 5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, pyruvic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+) -L-tartaric acid, thiocyanic acid, toluenesulfonic acid (e.g. p-toluenesulfonic acid), undecylenic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

A specific group of salts includes those formed from acetic, hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, acetic, propionic, butyric, malonic, glucuronic and lactobionic acids. Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulfonic, sebacic, stearic, succinic, and tartaric acids.

If the compound is anionic or has a functional group that can be anionic, a salt can be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions (e.g., Na)⁺And K⁺) Alkaline earth metal cations (e.g. Ca)²⁺And Mg²⁺) And other cations (e.g. A1)³⁺). Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH)₄ ⁺) And substituted ammonium ions (e.g. NH)₃R⁺、NH₂R₂ ⁺、NHR₃ ⁺、NR₄ ⁺)。

Examples of some suitable substituted ammonium ions are ammonium ions derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids (e.g., lysine and arginine). An example of a commonly used quaternary ammonium ion is N (CH)₃)₄ ⁺。

When the compounds of formula (I) contain amine functionality, these compounds may be reacted to form quaternary ammonium salts in a manner well known to the skilled person, for example by reaction with an alkylating agent. These quaternary ammonium salt compounds fall within the scope of formula (I). The compounds of formula (I) containing amine functions may also form N-oxides. The compounds of formula (I) containing amine functions mentioned herein also include N-oxides. When the compound contains several amine functional groups, one or more than one nitrogen atom may be oxidized to form an N-oxide. Specific examples of N-oxides are the N-oxygens of tertiary aminesA hydride or an N-oxide containing a nitrogen heterocyclic nitrogen atom. The corresponding amines can be treated with an oxidizing agent, such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acids), to form N-oxides, see, e.g., Jerry March,Advanced Organic Chemistry4 th edition, Wiley Interscience, pages. More specifically, the N-oxide can be prepared by the method of l.w. ready (Syn. Comm1977, 7, 509) -514) in which an amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA) in, for example, an inert solvent such as dichloromethane.

The compounds of the present invention may form solvates with, for example, water (i.e., hydrates) or common organic solvents. The term "solvate" as used herein means a physical association of a compound of the invention with one or more solvent molecules. Such physical associations include varying degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystalline lattice of a crystalline solid, the solvate will be able to dissociate. The term "solvate" is intended to include both solution phase and isolatable solvates. Non-limiting examples of suitable solvates include combinations of the compounds of the present invention with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine, and the like. The compounds of the invention exert their biological effect when in solution.

Solvates are well known in pharmaceutical chemistry. They will be important for the method of preparation of the substance (e.g. in relation to its purification), preservation of the substance (e.g. its stability) and ease of handling of the substance and often form part of the isolation or purification stage of the chemical synthesis. One skilled in the art can determine whether hydrates or other solvates are formed by the isolation conditions or purification conditions used to prepare a given compound by standard and long-term techniques. Examples of such techniques include thermogravimetric analysis (TGA), Differential Scanning Calorimetry (DSC), X-ray crystallography (e.g., single crystal X-ray crystallography or X-ray powder diffraction), and solid-state NMR (SS-NMR, also known as magic angle spin NMR or MAS-NMR). Such techniques are part of the standard analytical kit of the technical chemist as are NMR, IR, HPLC and MS. Alternatively, the skilled artisan may use crystallization conditions, including the amount of solvent required for a particular solvate, to deliberately form a solvate. Standard methods as described above can then be used to determine whether a solvate has formed. Formula (I) also includes any complex of a compound (e.g., an inclusion or clathrate with a compound (e.g., cyclodextrin), or a complex with a metal).

Furthermore, the compounds of the present invention may have one or more polymorphic (crystalline) or amorphous forms, and are likewise intended to be included within the scope of the present invention.

The compounds of formula (I) may exist in a number of different geometric isomers and tautomeric forms and reference to the compounds of formula (I) includes all such forms. For the avoidance of doubt, when a compound exists in one of several geometric or tautomeric forms and only one of them is specifically described or shown, all other forms are also included in formula (I). Other examples of tautomers include keto, enol, and enolate forms in, for example, the following tautomeric pairs: keto/enol (described below), imine/enamine, amide/imino alcohol, amidine/enediamine, nitroso/oxime, thione/enethiol, and nitro/acid nitro.

When a compound of formula (I) contains one or more chiral centers and two or more optical isomers may be present, reference to a compound of formula (I) includes all optical isomer forms thereof (e.g., enantiomers, epimers, and diastereomers), either as a single optical isomer, or as a mixture of two or more optical isomers (e.g., a racemic mixture), unless the context requires otherwise. Optical isomers can be characterized and identified by their optical activity (i.e., + and-isomers ordAndlisomer characterization and identification) or can be characterized in terms of its absolute stereochemistry using the "R and S" nomenclature developed by Cahn, Ingold and Prelog, see Jerry March,Advanced Organic Chemistry4 th edition, John Wiley&Sons, New York, 1992, page 109-; see also Cahn, Ingold and Prelog (1966),Angew. Chem. Int. Ed. Engl.,5, 385-415. Optical isomers can be separated by a variety of techniques, including chiral chromatography (chromatography on a chiral support), which techniques are well known to those skilled in the art. As an alternative to chiral chromatography, optical isomers can be separated as follows: diastereomeric salts with chiral acids such as (+) -tartaric acid, (-) -pyroglutamic acid, (-) -di-toluoyl-L-tartaric acid, (+) -mandelic acid, (-) -malic acid and (-) -camphorsulfonic acid, separation of the diastereomers by preferential crystallization, followed by dissociation of the salt to give the single enantiomer of the free base.

When the compound of formula (I) exists in two or more optically active forms, one enantiomer of a pair of enantiomers may exhibit advantages over the other enantiomer, for example in terms of biological activity. Thus, in some cases, it may be desirable to use a single one of the pair of enantiomers or a single one of a number of diastereomers as a therapeutic agent. Accordingly, the present invention provides compositions comprising a compound of formula (I) having one or more chiral centers, wherein at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound of formula (I) is present as a single optical isomer (e.g., an enantiomer or diastereomer). In a general embodiment, 99% or more (e.g., substantially all) of the total amount of the compound of formula (I) may be present as a single optical isomer (e.g., enantiomer or diastereoisomer). When a particular isomeric form (e.g., S configuration, or E isomer) is identified, this means that the isomeric form is substantially free of the other isomer, i.e., the isomeric form is present in at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., substantially all) of the total amount of the compound of the present invention.

Hereinbefore or hereinafter, whenever a compound comprises the following bond

This means that the compound is a single stereoisomer or a mixture of stereoisomers with unknown configuration.

Compounds of the present invention include compounds having one or more isotopic substitutions, where a particular element is referred toAll isotopes are included. For example, reference to hydrogen will¹H、²H, (D) and³h (T) are included. Likewise, references to carbon and oxygen, respectively, will¹²C、¹³C and¹⁴c and¹⁶o and¹⁸o is included. Isotopes may be radioactive or non-radioactive. In one embodiment of the invention, the compound does not contain a radioisotope. Preferably such compounds are for therapeutic use. However, in another embodiment, the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be used in diagnostic contexts.

Formula (I) also includes esters of compounds of formula (I) bearing a carboxylic acid group or a hydroxyl group such as carboxylic acid esters and acyloxy esters. In one embodiment of the invention, formula (I) includes within its scope esters of compounds of formula (I) bearing a hydroxyl group. In another embodiment of the present invention, formula (I) excludes within its scope esters of compounds of formula (I) bearing hydroxyl groups. An example (reverse ester) of an acyloxy group is represented by-OC (= O) R, where R is an acyloxy substituent, e.g., C_1-7Alkyl radical, C_3-20Heterocyclyl or C_5-20Aryl, preferably C_1-7An alkyl group. Specific examples of acyloxy include, but are not limited to, -OC (= O) CH₃(acetoxy), -OC (= O) CH₂CH₃、-OC(=O)C(CH₃)₃-OC (= O) Ph and-OC (= O) CH₂Ph。

For example, some prodrugs are esters of the active compound (e.g., physiologically acceptable esters that are metabolically labile). By "prodrug" is meant any compound that is converted, for example, in vivo to a biologically active compound of formula (I). During metabolism, the ester group is cleaved to yield the active drug. Such esters may be formed, for example, by esterification of any hydroxy groups on the parent compound, where appropriate, by first protecting any other reactive groups present in the parent compound, followed by deprotection if desired.

Examples of such metabolically labile esters include C_1-6Aminoalkyl groups [ e.g., aminoethyl; 2- (N, N-diethylamino) ethyl; 2- (4-morpholino) ethyl); and acyloxy-C_1-7Alkyl [ e.g., acyloxymethyl; acyloxyethyl radical(ii) a Pivaloyloxymethyl; acetoxymethyl; 1-acetoxyethyl; 1- (1-methoxy-1-methyl) ethyl-carbonyloxyethyl; 1- (benzoyloxy) ethyl; isopropoxy-carbonyloxymethyl; 1-isopropoxy-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl; 1-cyclohexyloxy-carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl; 1- (4-tetrahydropyranyloxy) carbonyloxyethyl; (4-tetrahydropyranyl) carbonyloxymethyl; and 1- (4-tetrahydropyranyl) carbonyloxyethyl]. Also, some prodrugs are activated enzymatically to give the active compound, or the compound when further subjected to a chemical reaction gives the active compound (e.g., antigen-directed enzyme prodrug therapy (ADEPT)), gene-directed enzyme prodrug therapy (GDEPT), ligand-directed enzyme prodrug therapy (LIDEPT)), and the like). For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.

Protein Tyrosine Kinase (PTK)

The compounds of the invention described herein inhibit or modulate the activity of certain tyrosine kinases and therefore the compounds will be useful in the treatment or prophylaxis, particularly the treatment of disease states or conditions mediated by such tyrosine kinases, particularly FGFR.

FGFR

The Fibroblast Growth Factor (FGF) family of Protein Tyrosine Kinase (PTK) receptors regulates a wide variety of physiological functions, including mitogenesis, wound healing, cell differentiation, and angiogenesis and development. Growth and proliferation of both normal and malignant cells are affected by changes in local concentrations of FGFs, which are extracellular signaling molecules that act as autocrine as well as paracrine factors. Autocrine FGF signaling may be particularly important in the progression of steroid hormone-dependent cancers to hormone-independent states. FGF and its receptor are expressed at high levels in several tissues and cell lines, and overexpression is thought to contribute to the malignant phenotype. Furthermore, several oncogenes are homologues of genes encoding growth factor receptors and there is a possibility of aberrant activation of FGF-dependent signaling in human pancreatic Cancer (Knight et al, Pharmacology and Therapeutics 2010125: 1 (105-.

The two prototype members are acidic fibroblast growth factor (aFGF or FGF1) and basic fibroblast growth factor (bFGF or FGF2), and at least twenty distinct FGF family members have been identified to date. The cellular response to FGF is conducted via four types of high affinity transmembrane protein tyrosine-kinase Fibroblast Growth Factor Receptors (FGFRs) (FGFR1-FGFR4) numbered 1-4.

Disruption of the FGFR1 pathway may affect tumor cell proliferation because in addition to proliferating endothelial cells, this kinase is activated in many tumor types. The over-expression and activation of FGFR1 in tumor-associated vasculature suggests a role for these molecules in tumor angiogenesis.

Recent studies have shown a relationship between FGFR1 expression and tumorigenicity in Classical Lobular Cancer (CLC). CLC caused 10-15% of all breast cancers, generally lacking expression of p53 and Her2 while maintaining estrogen receptor expression. Gene amplification of 8p12-p11.2 was demonstrated in about 50% of CLC cases and suggested to be associated with increased FGFR1 expression. Preliminary studies with siRNA directed against FGFR1 or small molecule inhibitors of this receptor indicate that cell lines with this expansion are particularly sensitive to inhibition of this signal transduction pathway. Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma that may result from abnormal proliferation and differentiation during skeletal myogenesis. FGFR1 was overexpressed in primary rhabdomyosarcoma tumors and was associated with hypomethylation of the 5' CpG island and aberrant expression of the AKT1, NOG and BMP4 genes. FGFR1 is also associated with lung squamous cell carcinoma, colorectal cancer, glioblastoma, astrocytoma, prostate cancer, small cell lung cancer, melanoma, head and neck cancer, thyroid cancer, uterine cancer.

Fibroblast growth factor receptor 2 has a high affinity for acidic and/or basic fibroblast growth factors as well as for keratinocyte growth factor ligands. Fibroblast growth factorSub-receptor 2 also mediates the potent osteogenic effect of FGF during osteoblast growth and differentiation. Mutations in fibroblast growth factor receptor 2 that result in complex functional changes are indicative of induction of abnormal ossification of cranial sutures (craniosynostosis), suggesting that FGFR signaling plays a major role in intramembranous bone formation. For example, in apert (ap) syndrome, characterized by premature cranial suture ossification, most cases are associated with point mutations responsible for gain of function in fibroblast growth factor receptor 2. In addition, mutation screening in syndrome-type craniosynostosis patients has shown that many recurrent episodesFGFR2Mutations are responsible for the severe feiffer syndrome (Pfeiffer syndrome). Specific mutations of FGFR2 include W290C, D321A, Y340C, C342R, C342S, C342W, N549H, K641R in FGFR 2.

Several serious abnormalities in human skeletal development, including Apert syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson skin twitch syndrome, and Fiffer syndrome, are associated with the occurrence of mutations in fibroblast growth factor receptor 2. Most, if not all, cases of fizeau syndrome (PS) are also caused by de novo mutations in the fibroblast growth factor receptor 2 gene, which have recently been shown to break one of the fundamental principles governing ligand specificity. That is, two mutant spliced forms of fibroblast growth factor receptors, FGFR2c and FGFR2b, have acquired the ability to bind to atypical FGF ligands and be activated by atypical FGF ligands. This loss of ligand specificity leads to aberrant signal transduction and suggests that the severe phenotype of these disease syndromes results from ectopic ligand-dependent activation of fibroblast growth factor receptor 2.

Genetic aberrations (e.g., chromosomal translocations or point mutations) in FGFR3 receptor tyrosine kinases result in ectopically expressed or deregulated, constitutively active FGFR3 receptors. This abnormality is associated with a subset of multiple myeloma and in bladder, hepatocellular, oral squamous cell, and cervical cancers. Therefore, FGFR3 inhibitors are useful for the treatment of multiple myeloma, bladder cancer, and cervical cancer. FGFR3 is also overexpressed in bladder cancer, particularly invasive bladder cancer. FGFR3 is often activated by mutations in urinary epithelial cancers (UC). Elevated expression was associated with mutations (85% of mutant tumors showed high levels of expression), but 42% of tumors without detectable mutations also showed overexpression, including many muscle-invasive tumors. FGFR3 is also associated with endometrial and thyroid cancer.

Overexpression of FGFR4 is associated with poor prognosis in both prostate and thyroid cancers. In addition, germline polymorphism (Gly388Arg) is associated with increased incidence of lung, breast, colon, liver (HCC) and prostate cancer. In addition, truncated forms of FGFR4 (including the kinase domain) were also found to be present in 40% of pituitary tumors, but absent from normal tissues. FGFR4 overexpression was observed in liver, colon and lung tumors. FGFR4 is implicated in colorectal and liver cancers, where expression of its ligand FGF19 is often elevated. FGFR4 is also associated with astrocytomas, rhabdomyosarcomas.

Fibrotic conditions are a serious medical problem caused by abnormal or excessive deposition of fibrous tissue. This occurs in a number of diseases including cirrhosis, glomerulonephritis, pulmonary fibrosis, systemic fibrosis, rheumatoid arthritis and the natural process of wound healing. The mechanism of pathological fibrosis is not well understood, but is thought to be caused by the action of various cytokines including Tumor Necrosis Factor (TNF), Fibroblast Growth Factor (FGF), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF β), which are involved in the proliferation of fibroblasts and the deposition of extracellular matrix proteins including collagen and fibronectin. This results in changes in tissue structure and function and subsequent pathology.

Numerous preclinical studies have demonstrated up-regulation of fibroblast growth factor in preclinical models of pulmonary fibrosis. TGF β 1 and PDGF are reported to be involved in the process of fibrogenesis, and other published works suggest that elevated FGF and the consequent increase in fibroblast proliferation may respond to elevated TGF β 1. The reported clinical effects of the anti-fibrotic drug pirfenidone suggest a potential therapeutic benefit of targeting the fibrotic mechanism in conditions such as Idiopathic Pulmonary Fibrosis (IPF). Idiopathic pulmonary fibrosis (also known as cryptogenic fibrosing alveolitis) is a progressive condition involving scarring of the lungs. The alveoli of the lungs are gradually replaced by fibrotic tissue, which becomes thicker, causing the irreversible loss of the tissue's ability to transport oxygen into the blood stream. Symptoms of this condition include shortness of breath, chronic dry cough, fatigue, chest pain, and loss of appetite leading to rapid weight loss. The condition is extremely severe with a 5 year mortality rate of about 50%.

Thus, compounds that inhibit FGFR may be used to provide a means of preventing growth or initiating apoptosis in tumors, particularly by inhibiting angiogenesis. It is therefore expected that the compounds will prove useful in the treatment or prevention of proliferative disorders, such as cancer. In particular tumors that have an activating mutant of a Receptor Tyrosine Kinase (RTK) or an upregulation of a receptor tyrosine kinase may be particularly sensitive to the inhibitor. Patients with activating mutants of any isoform of a particular RTK discussed herein may also find particular benefit in treatment with RTK inhibitors, such as patients with tumors (e.g., bladder or brain tumors) with FGFR3-TACC3 translocation.

Vascular Endothelial Growth Factor Receptor (VEGFR)

Chronic proliferative diseases are often accompanied by significant angiogenesis, which may contribute to or maintain an inflammatory and/or proliferative state, or cause tissue destruction through invasive proliferation of blood vessels.

Angiogenesis is commonly used to describe the development of new or replacement blood vessels, i.e., neovascularization. It is an essential physiological normal process whereby the vascular system is established in the embryo. Angiogenesis does not normally occur in most normal adult tissues except at the site of ovulation, menstruation and wound healing. However, many diseases are characterized by persistent and unregulated angiogenesis. For example, in arthritis, new capillaries invade the joints and destroy cartilage. In diabetes (and in many different eye diseases), new blood vessels invade the macula or retina or other ocular structures, and can lead to blindness. The atherosclerotic process is associated with angiogenesis. Tumor growth and metastasis have been found to be dependent on angiogenesis.

The recognition of the involvement of angiogenesis in major diseases is accompanied by research for the identification and development of angiogenesis inhibitors. These inhibitors are generally classified as responding to discrete targets in the angiogenic cascade (e.g., activation of endothelial cells by angiogenic signals; synthesis and release of degradative enzymes; endothelial cell migration; endothelial cell proliferation and capillary formation). Thus, angiogenesis occurs in many stages, and attempts are being made to find and develop compounds for blocking angiogenesis in these different stages.

There are publications that teach that angiogenesis inhibitors that act by different mechanisms are beneficial in diseases such as cancer and metastasis, ocular diseases, arthritis, and hemangiomas.

Vascular Endothelial Growth Factor (VEGF), a polypeptide, is mitogenic to endothelial cells in vitro and stimulates an angiogenic response in vivo. VEGF is also associated with inappropriate angiogenesis. VEGFR is a Protein Tyrosine Kinase (PTK). PTKs catalyze the phosphorylation of specific tyrosine residues in proteins involved in cellular function, thereby regulating cell growth, survival and differentiation.

Three PTK receptors for VEGF have been identified: VEGFR-1 (Flt-1); VEGFR-2 (Flk-1 or KDR) and VEGFR-3 (Flt-4). These receptors are involved in angiogenesis and in signal transduction. Of particular interest is VEGFR-2, which is a transmembrane receptor PTK expressed primarily in endothelial cells. Activation of VEGFR-2 by VEGF is a key step in the signal transduction pathway that initiates tumor angiogenesis. VEGF expression may be constitutive to tumor cells and may also be upregulated in response to certain stimuli. One such stimulus is hypoxia, where VEGF expression is upregulated in both tumor and related host tissues. The VEGF ligand activates VEGFR-2 by binding to its extracellular VEGF binding site. This results in receptor dimer formation of VEGFR and autophosphorylation of tyrosine residues at the intracellular kinase domain of VEGFR-2. The kinase domain functions to transfer phosphate from ATP to tyrosine residues, thereby providing a binding site for signal transduction proteins downstream of VEGFR-2, ultimately leading to initiation of angiogenesis.

Inhibition at the kinase domain binding site of VEGFR-2 blocks phosphorylation of tyrosine residues and acts to interfere with angiogenesis initiation.

Angiogenesis is a physiological process of new blood vessel formation mediated by various cytokines called angiogenic factors. Although the possible pathophysiological role in solid tumors has been extensively studied for over 30 years, enhanced angiogenesis in Chronic Lymphocytic Leukemia (CLL) and other hematologic malignancies has only recently been recognized. Increased levels of angiogenesis in the bone marrow and lymph nodes of CLL patients are demonstrated by various experimental methods. Although the role of angiogenesis in the pathophysiology of this disease remains to be fully elucidated, experimental data suggest that several angiogenic factors play a role in the disease process. Biomarkers of angiogenesis also indicate prognostic relevance in CLL. This suggests that VEGFR inhibitors may also be beneficial in leukemia (e.g., CLL) patients.

In order for the tumor mass to exceed a critical size, the associated vasculature must be formed. Targeting tumor vasculature has been proposed to limit tumor expansion and may be a useful cancer therapy. Observations of tumor growth indicate that small tumor masses can persist in tissues without any tumor-specific vasculature. Growth arrest in non-vascularized tumors is due to the effect of hypoxia in the center of the tumor. Recently, various pro-angiogenic and anti-angiogenic factors have been identified and the concept of an "angiogenesis switch" (a process in which the normal ratio of angiogenic stimulators and inhibitors in the tumor mass is disrupted leading to autonomous angiogenesis) has been developed. The angiogenic switch appears to be dominated by the same genetic alterations that drive malignant transformation (activation of oncogenes and loss of tumor suppressor genes). Several growth factors act as positive regulators of angiogenesis. The most important of these are Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF) and angiogenin. Thrombospondin (Tsp-1), angiostatin, and endostatin, among other proteins, act as negative regulators of angiogenesis.

Inhibition of VEGFR2, but not VEGFRl, significantly interfered with angiogenesis switching, persistent angiogenesis, and initial tumor growth in a mouse model. In advanced tumors, phenotypic tolerance to VEGFR2 blockade appeared as tumors regrown after the initial period of growth inhibition during treatment. This resistance to VEGF blockade includes reactivation of tumor angiogenesis, which is independent of VEGF and is associated with hypoxia-mediated induction of other pro-angiogenic factors, including members of the FGF family. These other pro-angiogenic signals are functionally involved in tumor revascularization and regrowth during escape, as FGF blockade impairs progression in the face of VEGF inhibition.

In a phase 2 study, there was evidence of normalization of glioblastoma blood vessels in patients treated with the pan-VEGF receptor tyrosine kinase inhibitor AZD 2171. MRI determination of blood vessel normalization in combination with circulating biomarkers provides an effective means of assessing the response to anti-angiogenic agents.

PDGFR

Malignant tumors are the result of uncontrolled cell proliferation. The delicate balance between growth promoting factors and growth inhibitory factors controls cell growth. In normal tissues, the production and activity of these factors results in the growth of differentiated cells in a controlled and regulated manner that maintains the normal integrity and function of the organ. Malignant cells evade this control; the natural balance is disturbed (by various mechanisms) and deregulated, abnormal cell growth occurs. A growth factor important in tumor development is Platelet Derived Growth Factor (PDGF), which comprises a family of peptide growth factors that signal through cell surface tyrosine kinase receptors (PDGFR) and stimulate various cellular functions including growth, proliferation and differentiation.

Advantages of Selective inhibitors

The development of FGFR kinase inhibitors with differentiated selectivity profiles provides a new opportunity to use these targeting agents in a subset of patients with diseases driven by FGFR dysregulation. Compounds that show reduced inhibition of other kinases (particularly VEGFR2 and PDGFR- β) offer the opportunity to have differential side effects or toxicity profiles and thus allow more effective treatment of these indications. Inhibitors of VEGFR2 and PDGFR- β are associated with toxicity, e.g., hypertension or edema, respectively. In the case of VEGFR2 inhibitors, this hypertension-causing effect is often dose-limiting, may appear to be contraindicated in certain patient populations, and requires clinical management.

Biological activity and therapeutic use

The compounds of the invention and subgroups thereof have Fibroblast Growth Factor Receptor (FGFR) inhibitory or modulating activity and/or Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitory or modulating activity and/or Platelet Derived Growth Factor Receptor (PDGFR) inhibitory or modulating activity and are useful in the prevention or treatment of a disease state or condition described herein. In addition, the compounds of the invention and subgroups thereof are useful in the prevention or treatment of kinase mediated diseases or conditions. Reference to preventing or treating a disease state or condition (e.g. cancer) includes within its scope the reduction or reduction of the incidence of cancer.

The term "modulation" as used herein when applied to kinase activity is intended to define changes in the level of biological activity of a protein kinase. Thus, modulation includes physiological changes that result in an increase or decrease in the activity of the relevant protein kinase. In the latter case, the modulation may be described as "suppression". Modulation may occur directly or indirectly, and may be mediated by any mechanism at any physiological level, including, for example, the level of gene expression (including, for example, transcriptional, translational and/or post-translational modifications), the level of expression of genes encoding regulatory elements that act directly or indirectly on the level of kinase activity. Thus, modulation may mean elevated/suppressed expression or over-or under-expression of the kinase, including gene amplification (i.e., multiple gene copies) and/or increased or decreased expression due to transcription, as well as over-or under-activation (including activation (inactivation)) and activation (including inactivation) of one or more protein kinases due to one or more mutations. The terms "modulated", "modulating" and "modulation" are also to be construed as such.

The term "mediated" as used herein, e.g., in conjunction with a kinase described herein (and applicable, e.g., to various physiological processes, diseases, conditions, therapies, treatments, or interventions), refers to the manipulation by limitations that allows the various processes, diseases, conditions, treatments, or interventions to which the term applies to those in which the kinase plays a biological role. Where the term applies to a disease, condition, or condition, the biological effect played by the kinase may be direct or indirect, and may be necessary and/or sufficient for symptomatic manifestation of the disease, condition, or condition (or cause or progression thereof). Thus, kinase activity (particularly abnormal levels of kinase activity, e.g., kinase overexpression) need not be a proximate cause of the disease, condition, or condition: rather, kinase-mediated diseases, conditions or conditions are contemplated to include those with multi-factorial etiology and in which the kinase is only partially involved in a complex process. Where the term applies to treatment, prophylaxis or intervention, the action played by the kinase may be direct or indirect and should be necessary and/or sufficient for the performance of the treatment, prophylaxis or the outcome of the intervention. Thus, a disease, condition or condition mediated by a kinase includes resistance to any particular cancer drug or treatment.

Thus, for example, the compounds of the invention may be used to reduce or reduce the incidence of cancer.

More specifically, compounds of formula (I) and subclasses thereof are inhibitors of FGFR. For example, the compounds of the invention have activity against FGFR1, FGFR2, FGFR3 and/or FGFR4, in particular FGFR selected from FGFR1, FGFR2 and FGFR 3; or inhibitors of the compounds of formula (I) and subclasses thereof, in particular FGFR 4.

Preferred compounds are compounds that inhibit one or more FGFR selected from FGFR1, FGFR2, FGFR3 and FGFR 4. Preferred compounds of the invention are IC₅₀Compounds with values less than 0.1 μ M.

The compounds of the invention also have activity against VEGFR.

Additionally, many of the compounds of the invention show selectivity for FGFR1, 2 and/or 3 and/or 4 as compared to VEGFR (particularly VEGFR2) and/or PDGFR, such compounds representing a preferred embodiment of the invention. The compounds in particular show selectivity relative to VEGFR 2. For example, IC of many of the compounds of the invention for FGFR1, 2 and/or 3 and/or 4₅₀The values are in IC for VEGFR (particularly VEGFR2) and/or PDGFR B₅₀1/10 and 1/100. In particular preferred compounds of the invention are at least 10 fold more active against or inhibiting FGFR, in particular FGFR1, FGFR2, FGFR3 and/or FGFR4 than against or inhibiting VEGFR 2. More preferably, the compounds of the present invention are directed against or inhibitFGFR, in particular FGFR1, FGFR2, FGFR3 and/or FGFR4 is at least 100-fold active against or inhibiting VEGFR 2. This can be determined using the methods described herein.

Due to their activity in modulating or inhibiting FGFR and/or VEGFR kinases, the compounds are useful to provide a means of preventing growth or inducing apoptosis in tumors, particularly by inhibiting angiogenesis. It is therefore expected that the compounds will prove useful in the treatment or prevention of proliferative disorders such as cancer. Furthermore, the compounds of the invention are useful in the treatment of diseases in which a proliferation, apoptosis or differentiation disorder is present.

In particular, patients with activating mutants of VEGFR or elevated levels of tumor and serum lactate dehydrogenase that are upregulated in VEGFR are particularly sensitive to the compounds of the present invention. Treatment with the compounds of the invention may also be found to be particularly beneficial to patients presenting with activating mutants of any isoform of the particular RTK discussed herein. For example, VEGFR is overexpressed in acute leukemia cells where clonal progenitor cells may express VEGFR. Furthermore, specific tumors having an activating mutant or upregulation or overexpression of any isoform of FGFR, such as FGFR1, FGFR2 or FGFR3 or FGFR4, may be particularly sensitive to the compounds of the invention, and thus treatment with the compounds of the invention has also been found to be particularly beneficial to the patients discussed herein who present the specific tumors. It may be preferred that the treatment involves or is directed to a mutated form of, for example, one of the receptor tyrosine kinases discussed herein. Tumors in which the mutation is present can be diagnosed using techniques known to those skilled in the art and described herein (e.g., RTPCR and FISH).

Examples of cancers that can be treated (or inhibited) include, but are not limited to, cancers such as bladder cancer, breast cancer, colon cancer (e.g., colorectal cancer, such as colon adenocarcinoma and colon adenoma), kidney cancer, urothelial cancer, uterine cancer, epidermal cancer, liver cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer and non-small cell lung cancer, lung squamous cell cancer), esophageal cancer, head and neck cancer, gallbladder cancer, ovarian cancer, pancreatic cancer (e.g., exocrine pancreatic cancer), gastric cancer, gastrointestinal cancer (also known as gastric cancer) (e.g., gastrointestinal stromal tumor), cervical cancer, endometrial cancer, thyroid cancer, prostate cancer, or skin cancer (e.g., squamous cell carcinoma or dermatofibrosarcoma protruberans); pituitary cancer, hematopoietic tumors of lymphoid lineage such as leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B cell lymphoma (e.g., diffuse large B cell lymphoma), T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, such as leukemia, acute and chronic myelogenous leukemias, chronic myelomonocytic leukemia (CMML), myeloproliferative diseases, myeloproliferative syndromes, myelodysplastic syndromes, or promyelocytic leukemia; multiple myeloma; thyroid follicular cancer; hepatocellular carcinoma, tumors of mesenchymal origin (e.g. Ewing's sarcoma), such as fibrosarcoma or rhabdomyosarcoma; tumors of the central or peripheral nervous system, such as astrocytomas, neuroblastomas, gliomas (e.g., glioblastoma multiforme), or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoacanthoma (keratocotanthhoma); thyroid follicular cancer; or Kaposi's sarcoma. In particular squamous cell carcinoma of the lung, breast cancer, colorectal cancer, glioblastoma, astrocytoma, prostate cancer, small cell lung cancer, melanoma, head and neck cancer, thyroid cancer, uterine cancer, gastric cancer, hepatocellular carcinoma, cervical cancer, multiple myeloma, bladder cancer, endometrial cancer, urothelial cancer, colon cancer, rhabdomyosarcoma, pituitary adenocarcinoma.

Examples of cancers that may be treated (or inhibited) include, but are not limited to, bladder cancer, urothelial cancer, metastatic urothelial cancer, urothelial cancer that cannot be resected following surgery, breast cancer, glioblastoma, lung cancer, non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma of the lung, small cell lung cancer, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, squamous cell carcinoma of the head and neck, gastric cancer, esophageal squamous cell carcinoma, adenocarcinoma of the esophagus, cholangiocarcinoma, hepatocellular carcinoma.

Certain cancers are resistant to treatment with specific drugs. This may be due to the tumor type or may result from treatment with the compound. In this regard, reference to multiple myeloma includes bortezomib-sensitive multiple myeloma or refractory multiple myeloma. Similarly, references to chronic myeloid leukemia include imatinib (imatanib) sensitive chronic myeloid leukemia and refractory chronic myeloid leukemia. Chronic myelogenous leukemia is also known as chronic myelogenous leukemia, or CML. Likewise, acute myeloid leukemia is also referred to as acute myeloblastic leukemia, acute myelogenous leukemia, acute non-lymphocytic leukemia or AML.

The compounds of the invention are also useful in the treatment of hematopoietic disorders (whether premalignant or stable) in which cell proliferation is abnormal, such as myeloproliferative disorders. Myeloproliferative disorders ("MPDs") are a class of myelopathies in which excessive cells are produced. They are involved and may develop myelodysplastic syndrome. Myeloproliferative diseases include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Another hematopoietic disorder is hypereosinophilia syndrome. T cell lymphoproliferative disorders include those derived from natural killer cells.

Furthermore, the compounds of the present invention are useful in gastrointestinal (also known as gastric) cancers, such as gastrointestinal stromal tumors. Gastrointestinal cancer refers to a malignant condition of the gastrointestinal tract including the esophagus, stomach, liver, biliary system, pancreas, intestine and anus.

Thus, in one embodiment, in the pharmaceutical composition, use or method of the invention for treating a disease or condition comprising abnormal cell growth, the disease or condition comprising abnormal cell growth is cancer.

Particular subclasses of cancer include multiple myeloma, bladder, cervical, prostate and thyroid cancer, lung, breast and colon cancer.

Other sub-classes of cancer include multiple myeloma, bladder cancer, hepatocellular carcinoma, oral squamous cell carcinoma, and cervical cancer.

The compounds of the invention having FGFR such as FGFR1 inhibitory activity are particularly useful in the treatment or prevention of breast cancer, in particular typical lobular cancer (CLC).

Because the compounds of the invention have FGFR4 activity, they may also be useful in the treatment of prostate or pituitary cancer, or they may be useful in the treatment of breast, lung, prostate, liver (HCC) or lung cancer.

In particular, the compounds of the invention as FGFR inhibitors are useful for the treatment of multiple myeloma, myeloproliferative disorders, endometrial, prostate, bladder, lung, ovarian, breast, gastric, colorectal, and oral squamous cell carcinoma.

Other subclasses of cancer are multiple myeloma, endometrial, bladder, cervical, prostate, lung, breast, colorectal and thyroid cancer.

In particular, the compounds of the invention are useful for the treatment of multiple myeloma (in particular multiple myeloma with a t (4;14) translocation or overexpression of FGFR 3), prostate cancer (hormone refractory prostate cancer), endometrial cancer (in particular endometrial tumors with an activation mutation of FGFR 2), and breast cancer (in particular lobular breast cancer).

In particular the compounds are useful for the treatment of lobular cancer, such as CLC (typical lobular cancer).

Because the compounds have activity against FGFR3, they are useful for the treatment of multiple myeloma and bladder cancer.

In particular, the compounds have activity against tumors with an FGFR3-TACC3 translocation, in particular bladder or brain tumors with an FGFR3-TACC3 translocation.

In particular, the compounds are useful for treating multiple myeloma which are t (4;14) translocation positive.

In one embodiment, the compounds are useful for treating sarcoma. In one embodiment, the compounds are useful for treating lung cancer, such as squamous cell carcinoma.

As the compounds have activity against FGFR2, they are useful for the treatment of endometrial, ovarian, gastric, hepatocellular, uterine, cervical and colorectal cancers. FGFR2 is also overexpressed in epithelial ovarian cancer, and thus the compounds of the invention are particularly useful in the treatment of ovarian cancer, e.g., epithelial ovarian cancer.

In one embodiment, the compounds are useful for the treatment of lung cancer, in particular NSCLC, squamous cell carcinoma, liver cancer, kidney cancer, breast cancer, colon cancer, colorectal cancer, prostate cancer.

The compounds of the present invention may also be used to treat tumors previously treated with VEGFR2 inhibitors or VEGFR2 antibodies (e.g., Avastin).

In particular, the compounds of the present invention are useful for the treatment of VEGFR 2-resistant tumors. VEGFR2 inhibitors and antibodies are useful for treating thyroid and renal cell carcinoma, and therefore the compounds of the invention are useful for treating VEGFR 2-tolerant thyroid and renal cell carcinoma.

The cancer may be a cancer that is sensitive to inhibition of any one or more of FGFR1, FGFR2, FGFR3, FGFR4, for example one or more FGFR selected from FGFR1, FGFR2 or FGFR 3.

Whether a particular cancer is one that is sensitive to FGFR or VEGFR signaling inhibition can be determined by means of cell growth assays as provided below or by means of methods as provided in the section entitled "diagnostic methods".

The compounds of the invention, in particular those having FGFR or VEGFR inhibitory activity, are particularly useful for the treatment or prevention of cancers of the type associated with or characterized by the presence of elevated levels of FGFR or VEGFR, such as those mentioned herein in the introductory part of the application.

The compounds of the invention are useful in the treatment of the adult population. The compounds of the invention are useful for treating the pediatric population.

It has been found that some FGFR inhibitors can be combined with other anticancer agents. For example, it may be beneficial to combine an inhibitor of induction of apoptosis with another agent that acts to regulate cell growth by a different mechanism, thereby treating two typical features of cancer development. Examples of such combinations are provided below.

The compounds of the invention are useful in the treatment of other conditions caused by proliferative disorders such as type II or non-insulin dependent diabetes mellitus, autoimmune diseases, head trauma, stroke, epilepsy, neurodegenerative diseases such as Alzheimer's disease, motoneuron disease, progressive supranuclear palsy, corticobasal degeneration and Pick's disease such as autoimmune diseases and neurodegenerative diseases.

One subset of disease states and conditions in which the compounds of the present invention may be useful consists of: inflammatory diseases, cardiovascular diseases and wound healing.

FGFR and VEGFR are also known to play a role in apoptosis, angiogenesis, proliferation, differentiation and transcription, and therefore the compounds of the present invention may also be used to treat the following non-cancer diseases: chronic inflammatory diseases such as systemic lupus erythematosus, autoimmune-mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, autoimmune diabetes, eczema hypersensitivity, asthma, COPD, rhinitis and upper respiratory diseases; cardiovascular diseases, such as cardiac hypertrophy, restenosis, atherosclerosis; neurodegenerative disorders such as Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy, and cerebellar degeneration; glomerulonephritis; myelodysplastic syndrome, ischemic injury-associated myocardial infarction, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxicity-induced liver disease or alcohol-related liver disease, blood diseases such as chronic anemia and aplastic anemia; degenerative diseases of the musculoskeletal system such as osteoporosis and arthritis, aspirin sensitive sinusitis, cystic fibrosis, multiple sclerosis, renal disease, and cancer pain.

Furthermore, mutations in FGFR2 are associated with several serious abnormalities in human skeletal development, and thus the compounds of the present invention are useful in the treatment of human skeletal dysplasia, including cranial suture disossification (craniosynostosis), apert (ap) syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson dermoconvex syndrome, and feverson syndrome.

Compounds of the invention having FGFR e.g. FGFR2 or FGFR3 inhibitory activity are particularly useful for the treatment or prevention of bone diseases. A particular skeletal disease is achondroplasia or lethal dwarfism (also known as lethal dysplasia).

The compounds of the invention having FGFR e.g. FGFR1, FGFR2 or FGFR3 inhibitory activity are particularly useful for the treatment or prevention of pathologies in which progressive fibrosis is a symptom. Fibrotic conditions that can be treated with the compounds of the invention include diseases that exhibit abnormal or excessive deposition of fibrous tissue, for example in the natural processes of cirrhosis, glomerulonephritis, pulmonary fibrosis, systemic fibrosis, rheumatoid arthritis and wound healing. The compounds of the invention are also particularly useful in the treatment of pulmonary fibrosis, particularly in idiopathic pulmonary fibrosis.

Overexpression and activation of FGFR and VEGFR in tumor-associated vasculature also suggests a role for the compounds of the invention in preventing and interfering with the initiation of tumor angiogenesis. The compounds of the invention are particularly useful in the treatment of cancer, metastasis, leukemias such as CLL, ocular diseases such as age-related macular degeneration, particularly wet age-related macular degeneration, ischemic proliferative retinopathies such as retinopathy of prematurity (ROP) and diabetic retinopathy, rheumatoid arthritis and hemangiomas.

The activity of the compounds of the invention as inhibitors of FGFR1-4, VEGFR and/or PDGFR A/B can be measured using the assays provided in the examples below and can be based on IC₅₀The values determine the activity level exhibited by a given compound. Preferred compounds of the invention are IC₅₀Compounds having a value of less than 1 μ Μ, more preferably less than 0.1 μ Μ.

The present invention provides compounds that have FGFR inhibitory or modulating activity and are useful in the prevention or treatment of disease states or conditions mediated by FGFR kinases.

In one embodiment, there is provided a compound as defined herein for use in therapy, for use as a medicament. In another embodiment, there is provided a compound as defined herein for use in the prophylaxis or treatment, in particular for use in the treatment of a disease state or condition mediated by a FGFR kinase.

Thus, for example, the compounds of the invention may be used to reduce or reduce the incidence of cancer. Thus, in another embodiment, there is provided a compound as defined herein for use in the prophylaxis or treatment, in particular for use in the treatment of cancer. In one embodiment, the compound as defined herein is for use in the prevention or treatment of FGFR-dependent cancer. In one embodiment, the compounds defined herein are for use in the prevention or treatment of cancer mediated by FGFR kinase.

Accordingly, the present invention provides, inter alia:

-a method for the prevention or treatment of a disease state or condition mediated by a FGFR kinase, which method comprises administering to a subject in need thereof a compound of formula (I) as defined herein.

-a method for the prophylaxis or treatment of a disease state or condition as described herein, which method comprises administering to a subject in need thereof a compound of formula (I) as defined herein.

-a method for the prevention or treatment of cancer, said method comprising administering to a subject in need thereof a compound of formula (I) as defined herein.

-a method for reducing or decreasing the incidence of a disease state or condition mediated by a FGFR kinase, which method comprises administering to a subject in need thereof a compound of formula (I) as defined herein.

-a method of inhibiting a FGFR kinase, which method comprises contacting the kinase with a kinase inhibiting compound of formula (I) as defined herein.

-methods of modulating a cellular process (e.g. cell division) by inhibiting the activity of an FGFR kinase using a compound of formula (I) as defined herein.

A compound of formula (I) as defined herein for use as a modulator of a cellular process (e.g. cell division) by inhibiting the activity of a FGFR kinase.

-a compound of formula (I) as defined herein for use in the prevention or treatment of cancer, in particular in the treatment of cancer.

-a compound of formula (I) as defined herein for use as an FGFR modulator (e.g. inhibitor).

-use of a compound of formula (I) as defined herein for the manufacture of a medicament for the prevention or treatment of a disease state or condition mediated by a FGFR kinase, said compound having formula (I) as defined herein.

-use of a compound of formula (I) as defined herein for the manufacture of a medicament for the prevention or treatment of a disease state or condition as described herein.

-the use of a compound of formula (I) as defined herein for the preparation of a medicament for the prophylaxis or treatment, in particular for the treatment of cancer.

-use of a compound of formula (I) as defined herein for the manufacture of a medicament for modulating (e.g. inhibiting) the activity of FGFR.

-the use of a compound of formula (I) as defined herein for the manufacture of a medicament for modulating a cellular process (e.g. cell division) by inhibiting the activity of an FGFR kinase.

-use of a compound of formula (I) as defined herein for the manufacture of a medicament for the prevention or treatment of a disease or condition characterised by upregulation of an FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR 4).

-use of a compound of formula (I) as defined herein for the manufacture of a medicament for the prevention or treatment of cancer characterized by upregulation of FGFR kinases (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR 4).

-use of a compound of formula (I) as defined herein for the manufacture of a medicament for the prevention or treatment of cancer in a patient selected from a subgroup with a genetic aberration of FGFR3 kinase.

-use of a compound of formula (I) as defined herein for the manufacture of a medicament for the prevention or treatment of cancer in a patient diagnosed to constitute part of a subpopulation having a genetic aberration of FGFR3 kinase.

-a method for the prevention or treatment of a disease or condition characterised by upregulation of an FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4), which method comprises administering a compound of formula (I) as defined herein.

-a method for reducing or decreasing the incidence of a disease or condition characterised by upregulation of an FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4), which method comprises administering a compound of formula (I) as defined herein.

-a method for preventing or treating (or reducing the incidence of) cancer in a patient having or suspected of having cancer; the method comprises (i) performing a diagnostic test on the patient to determine whether the patient has a genetic aberration of the FGFR3 gene; and (ii) when the patient does have said variant, subsequently administering to the patient a compound of formula (I) as defined herein having FGFR3 kinase inhibitory activity.

-a method for the prevention or treatment of (or reducing the incidence of) a disease state or condition characterised by upregulation of an FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR 4); the method comprises (I) subjecting the patient to a diagnostic test to detect a marker characteristic of the upregulation of an FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4) and (ii) when the diagnostic test indicates that the FGFR kinase is upregulated, concomitantly administering to the patient a compound of formula (I) as defined herein having FGFR kinase inhibitory activity.

In one embodiment, the disease mediated by a FGFR kinase is an oncology-related disease (e.g., cancer). In one embodiment, the disease mediated by a FGFR kinase is a non-oncology-related disease (e.g., any disease disclosed herein other than cancer). In one embodiment, the disease mediated by a FGFR kinase is a condition described herein. In one embodiment, the disease mediated by a FGFR kinase is a bone condition described herein. Specific abnormalities in human skeletal development include abnormal ossification of the cranial sutures (craniosynostosis), apert (ap) syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson dermatotwitch syndrome, feyfar syndrome, achondroplasia and lethal dwarfism (also known as lethal dysplasia).

Mutant kinase

Drug-resistant kinase mutations may occur in patient populations treated with kinase inhibitors. These moieties occur in regions of the protein that bind to or interact with the specific inhibitor used in the treatment. The mutation reduces or increases the ability of the inhibitor to bind to and inhibit the kinase. This may occur at any amino acid residue that interacts with the inhibitor or is important to support binding of the inhibitor to the target. Inhibitors that bind to the target kinase without interacting with the mutated amino acid residue are likely not affected by the mutation and will still be effective inhibitors of the enzyme.

Studies of gastric cancer patient samples indicated the presence of two mutations in FGFR 2-Serl 67Pro in exon IIIa and the splice site mutation 940-2A-G in exon IIIc. These mutations are identical to the germline activating mutations responsible for craniosynostosis syndrome and are observed in 13% of the primary gastric cancer tissues studied. Furthermore, an activating mutation of FGFR3 was observed in 5% of the patient samples tested, and overexpression of FGFR was correlated with poor prognosis in this patient group.

In addition, there are chromosomal translocations or point mutations observed in FGFR that result in a gain-of-performance, overexpressed, or constitutively active biological state.

The compounds of the invention are therefore particularly useful in cancers expressing a mutant molecular target (e.g. FGFR). Tumors having the mutation can be diagnosed using techniques known to those skilled in the art and described herein (e.g., RTPCR and FISH).

Mutations in conserved threonine residues at ATP-binding sites of FGFR have been shown to lead to inhibitor tolerance. Amino acid valine 561 has been mutated to methionine in FGFR1, which is equivalent to the mutations previously reported to be present in Abl (T315) and EGFR (T766), which have been shown to confer tolerance to selective inhibitors. The assay data for FGFR 1V 561M indicate that this mutation confers tolerance to tyrosine kinase inhibitors compared to the wild type.

Diagnostic method

Prior to administration of a compound of formula (I), the patient may be screened to determine whether the disease or condition from which the patient suffers or may suffer is one that is susceptible to treatment with a compound having activity against FGFR and/or VEGFR.

For example, a biological sample taken from a patient can be analyzed to determine whether a condition or disease (e.g., cancer) that the patient suffers from or is likely to suffer from is one characterized by a genetic abnormality or an abnormality in protein expression, wherein the abnormality results in upregulation of the level or activity of FGFR and/or VEGFR or in pathway sensitization of normal FGFR and/or VEGFR activity, or leads to upregulation of these growth factor signal transduction pathways (e.g., growth factor ligand level or growth factor ligand activity) or leads to upregulation of biochemical pathways downstream of FGFR and/or VEGFR activation.

Examples of such abnormalities that result in activation or sensitization of FGFR and/or VEGFR signals include loss or inhibition of apoptotic pathways, upregulation of receptors or ligands, or the presence of mutant variants of receptors or ligands (e.g., PTK variants). Tumors with mutants or up-regulation of FGFR1, FGFR2 or FGFR3 or FGFR4, in particular FGFR1 overexpression, or gain-of-function mutants of FGFR2 or FGFR3, are likely to be particularly sensitive to FGFR inhibitors.

For example, point mutations responsible for the gain of function of FGFR2 have been identified in a number of conditions. Especially the activating mutation of FGFR2 has been identified in 10% of endometrial tumors.

In addition, genetic aberrations (e.g., chromosomal translocations or point mutations) in the FGFR3 receptor tyrosine kinase that result in ectopically expressed or deregulated, constitutively active FGFR3 receptors have been identified and are associated with multiple myeloma, bladder cancer, and a subclass of cervical cancer. A specific mutation T674I of the PDGF receptor has been identified in imatinib-treated patients. Furthermore, gene amplification of 8p12-p11.2 was demonstrated in about 50% of lobular breast cancer (CLC) cases, suggesting a correlation with increased expression of FGFR 1. Preliminary studies using siRNA against FGFR1 or small molecule inhibitors of this receptor indicate that cell lines presenting such expansion are particularly sensitive to inhibition of this signal transduction pathway.

Alternatively, biological samples taken from patients may be analyzed for loss of negative modulators or inhibitors of FGFR or VEGFR. In this context, the term "loss" includes deletion of the gene encoding the modulator or inhibitor, truncation of the gene (e.g., by mutation), truncation of the gene transcript, or inactivation of the transcript (e.g., by point mutation) or sequestration by other gene products.

The term up-regulation includes elevated or over-expression, including gene amplification (i.e., multiple gene copies) and increased expression by transcription as well as over-activation and activation, including activation by mutation. Thus, a diagnostic assay may be performed on a patient to detect markers characteristic of FGFR and/or VEGFR upregulation. The term diagnosis includes screening. Markers include genetic markers including, for example, determining DNA composition to identify mutations in FGFR and/or VEGFR. The term markers also includes markers characteristic of FGFR and/or VEGFR upregulation, including enzyme activity, enzyme levels, enzyme status (e.g., phosphorylated or not), and mRNA levels of the foregoing proteins.

Diagnostic tests and screens are usually performed with biological samples selected from tumor biopsy samples, blood samples (isolation and enrichment of shed tumor cells), stool biopsy samples, sputum, chromosome analysis, pleural fluid, peritoneal fluid, oral smears, biopsy samples or urine.

Methods for the identification and analysis of mutations and upregulation of proteins are known to those skilled in the art. Screening methods may include, but are not limited to, standard methods, such as reverse transcriptase polymerase chain reaction (RT-PCR) or in situ hybridization, such as Fluorescence In Situ Hybridization (FISH), for example.

Identifying an individual carrying FGFR and/or VEGFR mutations can refer to a patient that is particularly suited for treatment with an FGFR and/or VEGFR inhibitor. Prior to treatment, tumors can be preferentially screened for the presence of FGFR and/or VEGFR variants. Screening methods may typically include direct sequencing, oligonucleotide microarray analysis, or mutant specific antibodies. In addition, tumors having the mutations can be diagnosed using techniques known to those skilled in the art and described herein (e.g., RT-PCR and FISH).

Furthermore, mutant forms of, for example, FGFR or VEGFR2 can be identified by direct sequencing of, for example, tumor biopsy samples using PCR and by direct sequencing of PCR products as described above. The skilled person will appreciate that all such well known techniques for detecting overexpression, activation or mutation of the above proteins may be applicable to the present invention.

In screening by RT-PCR, mRNA levels in tumors are assessed by generating cDNA copies of the mRNA, followed by amplification of the cDNA by PCR. PCR amplification methods, primer selection and amplification conditions are known to those skilled in the art. Current Protocols in molecular biology, John Wiley & Sons inc.; or Innis, M.A. et al, eds (1990) PCR Protocols: a guide to methods and applications, Academic Press, San Diego, for nucleic acid manipulation and PCR. Reactions and manipulations involving nucleic acid technology are also described in Sambrook et al (2001), 3 rd edition, Molecular Cloning: A Laboratory Manual, Cold spring harbor Laboratory Press. Alternatively, commercially available RT-PCR kits (e.g., Roche Molecular Biochemicals) or methods as provided in U.S. patents 4,666,828, 4,683,202, 4,801,531, 5,192,659, 5,272,057, 5,882,864, and 6,218,529, and incorporated herein by reference, can be used. An example of an in situ hybridization technique for assessing mRNA expression is Fluorescence In Situ Hybridization (FISH) (see anger (1987) meth. enzymol., 152: 649).

In general, in situ hybridization comprises the following major steps: (I) fixing the tissue to be analyzed; (2) sample prehybridization treatment to improve accessibility of target nucleic acids and reduce non-specific binding; (3) hybridizing the mixture of nucleic acids to nucleic acids in a biological structure or tissue; (4) washing after hybridization to remove nucleic acid fragments that are not bound during hybridization, and (5) detecting the hybridized nucleic acid fragments. Probes used in such applications are typically labeled with, for example, radioisotopes or fluorescent reporters. Preferred probes are long enough, e.g., about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to be capable of specifically hybridizing to a target nucleic acid under stringent conditions. Standard Methods for performing FISH are described In Ausubel, F.M. et al, eds (2004) Current Protocols In Molecular Biology, John Wiley & Sons Inc and Fluorescence In Situ Hybridization of John M.S. Bartlet Technical Overview, by Molecular diagnostics of Cancer, Methods and Protocols, 2 nd edition, ISBN: 1-59259-760-2, 2004 month 3, page 077-088; a series of publications, Methods in Molecular Medicine.

(DePrimo et al (2003),BMC Cancer3:3) methods for gene expression profiling are described. Briefly, the scheme is as follows: double-stranded cDNA was synthesized from total RNA using (dT)24 oligomers that prime first-strand cDNA synthesis, followed by synthesis of second-strand cDNA using random hexamer primers. Double-stranded cDNA was used as template for in vitro transcription of cRNA using biotinylated ribonucleotides. cRNA was chemically fragmented and then hybridized on Human genomic Arrays (Human Genome Arrays) overnight according to the protocol described by Affymetrix (Santa Clara, CA, USA).

Alternatively, protein products expressed from mRNA can be assayed by immunohistochemistry of tumor samples, solid phase immunoassays with microtiter plates, Western blotting, two-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry, and other methods known in the art for the detection of specific proteins. The detection method may comprise the use of site-specific antibodies. The skilled artisan will appreciate that all such well known techniques for detecting upregulation of FGFR and/or VEGFR or detecting FGFR and/or VEGFR variants or mutants are applicable to the present invention.

Abnormal levels of proteins, such as FGFR or VEGFR, can be measured using standard enzyme assays, such as those described herein. Activation or overexpression can also be detected in tissue samples, for example in tumor tissue. Tyrosine kinase activity is measured by using an assay, for example from Chemicon International. The target tyrosine kinase is immunoprecipitated from the sample lysate and its activity is measured.

An alternative method for measuring overexpression or activation of FGFR or VEGFR (including isoforms thereof) comprises measuring microvascular density. This can be measured, for example, using the method described by Orre and Rogers (Int J Cancer (1999), 84(2) 101-8). Assays also include the use of markers, for example in the case of VEGFR, these include CD31, CD34, and CD 105.

Thus, all of these techniques can also be used to identify tumors that are particularly suitable for treatment with the compounds of the present invention.

The compounds of the invention are particularly useful for treating patients with mutant FGFR. A G697C mutation in FGFR3 that results in constitutive activation of kinase activity was observed in 62% of oral squamous cell carcinomas. Activating mutations of FGFR3 have also been identified in bladder cancer cases. These mutations are 6 mutations with varying degrees of morbidity (prediction): R248C, S249C, G372C, S373C, Y375C, K652Q. In addition, the Gly388Arg polymorphism in FGFR4 was found to be associated with increased incidence and invasiveness of prostate, colon, lung, liver (HCC) and breast cancer. The compounds of the invention are particularly useful for treating patients having an FGFR3-TACC3 translocation.

Thus, in a further aspect, the invention includes the use of a compound of the invention for the manufacture of a medicament for the treatment or prevention of a disease state or condition in a patient who has been screened for and determined to have, or to be at risk of having, a disease or condition susceptible to treatment with a compound having activity against FGFR.

Specific mutations in the screened patients included the G697C, R248C, S249C, G372C, S373C, Y375C, K652Q mutations in FGFR3 and the Gly388Arg polymorphism in FGFR 4.

In another aspect, the invention includes a compound of the invention for use in the prevention or treatment of cancer in a patient selected from a subgroup having a FGFR gene variant (e.g., the G697C mutation in FGFR3 and the Gly388Arg polymorphism in FGFR 4).

MRI assays of vascular normalization in combination with circulating biomarkers (circulating progenitor cells (CPCs), CECs, SDF1, and FGF2), e.g., using MRI gradient echoes, spin echoes, and contrast enhancement to measure blood volume, relative vessel size, and vascular permeability, can also be used to identify VEGFR 2-resistant tumors treated with the compounds of the present invention.

Pharmaceutical compositions and combinations

In view of their useful pharmacological properties, the subject compounds may be formulated in a variety of pharmaceutical forms for administration purposes.

In one embodiment, a pharmaceutical composition (e.g., formulation) comprises at least one active compound of the invention in combination with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

To prepare the pharmaceutical compositions of the present invention, an effective amount of a compound of the present invention as an active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. The pharmaceutical composition may be in any form suitable for oral, parenteral, topical, intranasal, ocular, otic, rectal, intravaginal or transdermal administration. These pharmaceutical compositions are suitably in unit dosage form preferably suitable for oral, rectal, transdermal or parenteral administration by injection. For example, in the preparation of compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or in the case of powders, pills, capsules and tablets, solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like.

Because of their ease of administration, tablets and capsules represent the most advantageous oral unit dosage form in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least to a large extent, although other ingredients, for example to aid solubility, may also be included. For example, injectable solutions may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In compositions suitable for transdermal administration, the carrier optionally comprises a penetration enhancer and/or a suitable wetting agent, optionally in combination with a minor proportion of suitable additives of any nature which do not cause significant deleterious effects on the skin. The additives may facilitate administration to the skin and/or may aid in the preparation of the desired composition. These compositions can be administered in various ways, for example as a transdermal patch, as a spot-on formulation, as an ointment. It is particularly advantageous to formulate the above pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein the specification and claims, unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with a required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets (powder packets), wafers, injectable solutions or suspensions, teaspoonfuls, tablespoonfuls, and the like, as well as segregated multiples thereof (segregated multiples).

It is particularly advantageous to formulate the above pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein the specification and claims, unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with a required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powders in bags, wafers, injectable solutions or suspensions, teaspoonful, tablespoonful and the like, and segregated multiples thereof.

The compounds of the present invention are administered in an amount sufficient to exert their anti-tumor activity.

One skilled in the art can readily determine the effective amount from the test results provided below. In general, a therapeutically effective amount of 0.005 mg/kg to 100 mg/kg body weight, in particular 0.005 mg/kg to 10 mg/kg body weight, is contemplated. It may be appropriate to administer the required dose in 1,2, 3, 4 or more divided doses at appropriate intervals throughout the day. The divided doses may be formulated in unit dosage forms, for example containing from 0.5 to 500mg, especially from 1mg to 500mg, more especially from 10 mg to 500mg, of active ingredient per unit dosage form.

Depending on the mode of administration, the pharmaceutical composition preferably comprises 0.05 to 99% by weight, more preferably 0.1 to 70% by weight, even more preferably 0.1 to 50% by weight of a compound of the invention and 1 to 99.95% by weight, more preferably 30 to 99.9% by weight, even more preferably 50 to 99.9% by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.

As a further aspect of the invention, a combination of a compound of the invention with another anti-cancer agent is contemplated, especially for use as a medicament, more specifically for the treatment of cancer or related diseases.

For the treatment of the above conditions, the compounds of the invention may advantageously be used in combination with one or more other agents, more particularly with other anticancer drugs or adjuvants, in cancer therapy. Examples of anti-cancer drugs or adjuvants (carriers in this therapy) include, but are not limited to:

-platinum coordination compounds, such as cisplatin optionally in combination with amifostine, carboplatin or oxaliplatin;

taxanes, such as paclitaxel, paclitaxel protein-binding particles (Abraxane ™ or docetaxel;

topoisomerase I inhibitors, for example camptothecin compounds, such as irinotecan, SN-38, topotecan hydrochloride;

topoisomerase II inhibitors, such as anti-tumour epipodophyllotoxins or podophyllotoxin derivatives, such as etoposide, etoposide phosphate or teniposide;

anti-tumour vinca alkaloids, such as vinblastine, vincristine or vinorelbine;

antineoplastic nucleoside derivatives, such as 5-fluorouracil, leucovorin, gemcitabine hydrochloride, capecitabine, cladribine, fludarabine, nelarabine;

alkylating agents, such as nitrogen mustards or nitrosoureas, such as cyclophosphamide, chlorambucil, carmustine, thiotepa, melphalan (melphalan), lomustine, altretamine, busulfan, dacarbazine, estramustine, ifosfamide optionally in combination with mesna, pipobroman, procarbazine, streptozocin, temozolomide (telozolomide), uracil;

anti-tumour anthracycline derivatives, such as daunorubicin, doxorubicin, doxil, idarubicin, mitoxantrone, epirubicin hydrochloride, valrubicin, optionally in combination with dexrazoxane;

molecules targeting IGF-1 receptors, such as picropodophilin (picropodophilin);

-tetracaine derivatives, such as tetrarcin a;

glucocorticoids, such as prednisone;

antibodies, such as trastuzumab (HER2 antibody), rituximab (CD20 antibody), gemtuzumab ozolomide, cetuximab, pertuzumab, bevacizumab, alemtuzumab, eculizumab, ibritumomab tiuxetan, nomuzumab, panitumumab, tositumomab, CNTO 328;

-an estrogen receptor antagonist or a selective estrogen receptor modulator or an estrogen synthesis inhibitor, such as tamoxifen, fulvestrant, toremifene, droloxifene, faslodex, raloxifene or letrozole;

aromatase inhibitors, such as exemestane, anastrozole, letrozole, testolactone and vorozole;

differentiating agents, such as retinoids, vitamin D or retinoic acid and Retinoic Acid Metabolism Blockers (RAMBA), such as isotretinoin (accutane);

-DNA methyltransferase inhibitors, such as azacytidine or decitabine;

antifolates, such as pemetrexed disodium (premetrexed disodium);

antibiotics, such as actinomycin D (antinomycin D), bleomycin, mitomycin C, dactinomycin, carminomycin, daunomycin, levamisole, plicamycin, mithramycin;

antimetabolites, such as clofarabine, aminopterin, cytarabine or methotrexate, azacitidine, cytarabine (cytarabine), floxuridine, pentostatin, thioguanine;

apoptosis inducers and anti-angiogenic agents, such as Bcl-2 inhibitors, e.g. YC 137, BH 312, ABT 737, gossypol, HA 14-1, TW 37 or decanoic acid;

-tubulin-binding agents, such as combretastatin, colchicine or nocodazole;

kinase inhibitors (e.g. EGFR (epidermal growth factor receptor) inhibitors, MTKI (multi-target kinase inhibitors), mTOR inhibitors, cmet inhibitors), such as flavopiridol (flavoperidol), imatinib mesylate, erlotinib, gefitinib, dasatinib, lapatinib ditosylate, sorafenib, sunitinib maleate, temsirolimus, 6- { difluoro [6- (1-methyl-1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinoline or a pharmaceutically acceptable salt thereof, 6- [ difluoro (6-pyridin-4-yl [1,2,4] triazolo [4,3-b ] pyridazin-3-yl) methyl ] quinoline or a pharmaceutically acceptable salt thereof;

-farnesyltransferase inhibitors, such as tipifarnib;

-Histone Deacetylase (HDAC) inhibitors, such as sodium butyrate, suberoylanilide hydroxamic acid (SAHA), depsipeptides (FR 901228), NVP-LAQ824, R306465, JNJ-26481585, trichostatin a, vorinostat;

ubiquitin-proteasome pathway inhibitors, such as PS-341, mln.41 or bortezomib;

- Yondelis；

telomerase inhibitors, such as Telomestatin;

matrix metalloproteinase inhibitors, such as batimastat, marimastat, prinostat (prinostat) or metamastat (metastat);

recombinant interleukins, such as aldesleukin, denileukin-toxin linker (denileukindiftitox), interferon alpha 2a, interferon alpha 2b, pegylated interferon alpha 2 b;

-a MAPK inhibitor;

retinoids, such as alitretinoin, bexarotene, tretinoin (tretinoin);

-arsenic trioxide;

-an asparaginase enzyme;

steroids, such as drostandrosterone propionate, megestrol acetate, nandrolone (nandrolone decanoate, nandrolone phenylpropionate), dexamethasone;

-gonadotropin releasing hormone agonists or antagonists, such as abarelix, goserelin acetate, histrelin acetate, leuprolide acetate;

-thalidomide, lenalidomide;

-mercaptopurine, mitotane, pamidronate, peregase, pemetrexed, labyrinase;

-BH3 mimetics, such as ABT-737;

MEK inhibitors, such as PD98059, AZD6244, CI-1040;

-colony stimulating factor analogues, such as filgrastim, pegfilgrastim, sargrastim; erythropoietin or an analog thereof (e.g., dabigatran α); interleukin 11; an opper interleukin; zoledronic acid salts, zoledronic acid; fentanyl; a bisphosphonate; (ii) palifermin;

-steroidal cytochrome P45017 α -hydroxylase-17, 20-lyase inhibitors (CYP17), such as abiraterone, abiraterone acetate.

In one embodiment, the invention relates to a combination of a compound of formula (I), a pharmaceutically acceptable salt thereof or solvate thereof or any subclass thereof and examples, and 6- { difluoro [6- (1-methyl-1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinoline or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a combination of a compound of formula (I), a pharmaceutically acceptable salt thereof or solvate thereof or any subclass thereof and examples, and 6- [ difluoro (6-pyridin-4-yl [1,2,4] triazolo [4,3-b ] pyridazin-3-yl) methyl ] quinoline or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of formula (I), a pharmaceutically acceptable salt thereof or solvate thereof or any subclass thereof and examples, and 6- { difluoro [6- (1-methyl-1H-pyrazol-4-yl) [1,2,4] triazolo [4,3-b ] pyridazin-3-yl ] methyl } quinoline or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of formula (I), a pharmaceutically acceptable salt thereof or solvate thereof or any subclass thereof and examples, and 6- [ difluoro (6-pyridin-4-yl [1,2,4] triazolo [4,3-b ] pyridazin-3-yl) methyl ] quinoline or a pharmaceutically acceptable salt thereof.

The compounds of the invention also have therapeutic applications in sensitizing tumor cells for radiation therapy and chemotherapy.

The compounds of the invention may therefore be used as "radiosensitizers" and/or "chemosensitizers", or may be administered in combination with another "radiosensitizer" and/or "chemosensitizer".

The term "radiosensitizer," as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to an animal in a therapeutically effective amount to increase the sensitivity of cells to ionizing radiation and/or to promote the treatment of an ionizing radiation treatable condition.

The term "chemosensitizer", as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to an animal in a therapeutically effective amount to increase the sensitivity of the cell to chemotherapy and/or to promote the treatment of a disease treatable by chemotherapy.

Several mechanisms of the mode of action of radiosensitizers have been proposed in the literature, including: hypoxic cell radiosensitizers (e.g., 2-nitroimidazole compounds and benzotriazine dioxide compounds) mimic oxygen or behave like bioreductive agents under hypoxic conditions; non-hypoxic cell radiosensitizers (e.g., halogenated pyrimidines) can be analogs of DNA bases and preferentially incorporate into the DNA of cancer cells and thereby promote radiation-induced DNA molecule breakage and/or impede normal DNA repair mechanisms; various other possible mechanisms of action of radiosensitizers in the treatment of disease have been proposed.

Many cancer treatment regimens currently employ radiosensitizers in combination with X-ray radiation. Examples of X-ray activated radiosensitizers include, but are not limited to: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimozole, mitomycin C, RSU 1069, SR 4233, Ε O9, RB 6145, niacinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin and therapeutically effective analogs and derivatives of the radiosensitizers.

Photodynamic therapy (PDT) of cancer utilizes visible light as a radiation activator for the sensitizer. Examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, photosensitizers, benzoporphyrin derivatives, tin protoporphyrin, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine and therapeutically effective analogs and derivatives of the photodynamic radiosensitizers.

Radiosensitizers can be administered in combination with a therapeutically effective amount of one or more other compounds, including but not limited to: a compound that promotes the incorporation of a radiosensitizer into a target cell; compounds that control the flow of therapeutic agents, nutrients and/or oxygen to target cells; chemotherapeutic agents that act on tumors with or without other radiation; or other therapeutically effective compounds for the treatment of cancer or other diseases.

The chemosensitizer may be administered in combination with a therapeutically effective amount of one or more other compounds, including but not limited to: a compound that promotes the incorporation of a chemosensitizer into a target cell; compounds that control the flow of therapeutic agents, nutrients and/or oxygen to target cells; chemotherapeutic agents that act on tumors or other therapeutically effective compounds for the treatment of cancer or other diseases. It has been found that calcium antagonists (e.g., verapamil) can be used in combination with anti-neoplastic agents to establish chemosensitivity in tumor cells that are resistant to recognized chemotherapeutic agents and to enhance the efficacy of such compounds in drug-sensitive malignancies.

The components of the combination of the invention, i.e. the one or more other agents and the compound of the invention, may be formulated in various pharmaceutical forms for administration purposes, taking into account their useful pharmacological properties. These components may be formulated separately in separate pharmaceutical compositions or in a single pharmaceutical composition containing all of the components.

Accordingly, the present invention also relates to pharmaceutical compositions comprising one or more additional pharmaceutical agents and a compound of the present invention, together with a pharmaceutically acceptable carrier.

The invention also relates to the use of a combination of the invention for the preparation of a pharmaceutical composition for inhibiting the growth of tumor cells.

The invention also relates to products containing a compound of the invention as a first active ingredient and one or more anticancer agents as further active ingredients, as a combined preparation for simultaneous, separate or sequential use in the treatment of a patient suffering from cancer.

One or more additional agents and a compound of the invention may be administered simultaneously (e.g., in separate compositions or a single composition) or sequentially in either order. In the latter case, the two or more compounds are administered over a period of time and in an amount and manner sufficient to ensure that a beneficial or synergistic effect is achieved. It will be appreciated that the preferred method and sequence of administration and the respective dosages and schedules of the individual components of the combination will depend on the particular other agent to be administered and the compound of the invention, its route of administration, the particular tumour being treated and the particular host being treated. Optimal methods and sequences of administration, as well as dosages and schedules, can be readily determined by those skilled in the art using routine methods and in light of the information provided herein.

The weight ratio of the compound of the invention to one or more other anticancer agents when administered as a combination can be determined by one skilled in the art. As is well known to those skilled in the art, the ratio and exact dosage and frequency of administration will depend upon the particular compound of the invention and other anticancer agent employed, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, diet, time and general physical condition of the particular patient, the mode of administration, and other drugs the individual may be using. Furthermore, it will be apparent that the effective daily amount may be reduced or increased based on the response of the subject being treated and/or based on the evaluation of the physician prescribing the compounds of the instant invention. The specific weight ratio of the compound of formula (I) of the present invention to another anticancer agent may range from 1/10-10/1, more particularly 1/5-5/1, even more particularly 1/3-3/1.

The platinum coordination compound is advantageously present in an amount of 1 to 500 mg/square meter (mg/m) per course of treatment²) Body surface area, e.g. 50-400mg/m²Is administered, in particular about 75 mg/m for cisplatin²In a dose of about 300mg/m for carboplatin²。

The taxane compound is advantageously administered in an amount of 50-400mg/m per course of treatment²) Body surface area, e.g. 75-250 mg/m²Is about 175-250 mg/m, especially for paclitaxel²The dose is about 75-150 mg/m for docetaxel²。

The camptothecin compound is advantageously administered in an amount of 0.1-400 mg/m per course of treatment²) Body surface area, e.g. 1-300mg/m²Is administered, in particular about 100-350 mg/m for irinotecan²Dosage, for topotecan, is about 1-2mg/m²。

The antitumor podophyllotoxin derivative is advantageously administered at 30-300 mg/m per course of treatment (mg/m)²) Body surface area, e.g. 50-250mg/m²Is administered in a dosage of about 35-100 mg/m, particularly for etoposide²The dosage is about 50-250mg/m for teniposide²。

The antitumor alkaloid is preferably 2-30 mg/m (mg/m) per treatment course²) The dosage of the body surface area is about 3-12 mg/m, especially for vinblastine²In a dose of about 1-2mg/m for vincristine²In a dose of about 10-30 mg/m for vinorelbine²The dosage of (a).

The antitumor nucleoside derivative is advantageously used at 200-2500 mg/m (mg/m) per course of treatment²) Body surface area, e.g. 700-1500 mg/m²Is 200mg/m, in particular for 5-FU²The dose of (a) is about 800-1200 mg/m for gemcitabine²The dose of (a) is about 1000-2500 mg/m for capecitabine²。

Alkylating agents (e.g., nitrogen mustards or nitrosoureas) are advantageously used at 100-500 mg/square meter (mg/m) per course of treatment²) Body surface area, e.g. 120-200 mg/m²Is about 100 mg/m, especially for cyclophosphamide²The dosage of (A) is about 0.1-0.2 mg/kg for chlorambucil and about 150-200mg/m for carmustine²In a dose of about 100-150 mg/m for lomustine²The dosage of (a).

The antitumor anthracycline derivative is advantageously administered in an amount of 10-75 mg/m per course of treatment (mg/m)²) Body surface area, e.g. 15-60 mg/m²Is administered, in particular about 40-75 mg/m for doxorubicin²In a dose of about 25-45mg/m for daunorubicin²In a dose of about 10-15 mg/m for idarubicin²The dosage of (a).

The antiestrogens are advantageously administered in a dosage of about 1-100 mg per day, depending on the particular agent and condition to be treated. Tamoxifen is advantageously administered orally at a dose of 5-50 mg, preferably 10-20 mg, twice daily, continuing the treatment for a sufficient time to achieve and maintain the therapeutic effect. Toremifene is advantageously administered orally once daily at a dose of about 60mg, and the treatment is continued for a sufficient time to achieve and maintain a therapeutic effect. Anastrozole is advantageously administered orally once daily at a dose of about 1 mg. Droloxifene is advantageously administered orally once daily at a dose of about 20-100 mg. Raloxifene is advantageously administered orally once daily at a dose of about 60 mg. Exemestane is advantageously administered orally once daily in a dose of about 25 mg.

The antibody is advantageously present at about 1-5 mg/square meter (mg/m)²) The body surface area is dosed or, if different, administered as known in the art. Trastuzumab is advantageously administered at 1-5 mg/m per course of treatment (mg/m)²) Body surface area, in particular 2-4mg/m²The dosage of (a).

These doses may be administered, for example, once, twice or more per course of treatment, which may be repeated, for example, every 7, 14, 21 or 28 days.

The compounds of formula (I), their pharmaceutically acceptable addition salts (especially the pharmaceutically acceptable acid addition salts) and stereoisomeric forms thereof may have valuable diagnostic properties, since they may be used to detect or identify the formation of complexes between the marker compounds and other molecules, peptides, proteins, enzymes or receptors.

The detection or identification method may use a compound labeled with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like. Examples of radioactive isotopes include¹²⁵I、¹³¹I、³H and¹⁴C. enzymes are typically detected by conjugation to a suitable substrate and thereby catalyzing a detectable reaction. Examples thereof include, for example, β -galactosidase, β -glucosidase, alkaline phosphatase, peroxidase, and malate dehydrogenase, preferably horseradish peroxidase. Luminescent substances include, for example, luminol derivatives, luciferin, aequorin, and luciferase.

A biological sample may be defined as a body tissue or a body fluid. Examples of body fluids are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva, etc.

General synthetic route

The following examples illustrate the invention, but are merely examples and are not intended to limit the scope of the claims in any way.

Experimental part

Hereinafter, the term "DCM" meansDichloromethane, "Me" means methyl, "Et" means ethyl, "MeOH" means methanol, "DMF" means dimethylformamide, "Et₂O "means diethyl ether," EtOAc "means ethyl acetate," CAN "means acetonitrile," H₂O "means water," THF "means tetrahydrofuran," MgSO₄"means magnesium sulfate," NH₄OH "means ammonium hydroxide," K₂CO₃"means dipotassium carbonate," MgCl₂"means magnesium chloride," iPrNH₂"means isopropylamine," NH₄HCO₃"means ammonium bicarbonate," DMSO "means dimethyl sulfoxide," EDTA "means ethylenediaminetetraacetic acid," NADP "means nicotinamide adenine dinucleotide phosphate," SFC "means supercritical liquid chromatography," MP "means melting point.

A. Preparation of intermediates

Intermediate 1 or N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1-methyl-1H-pyrazol-4-yl) quinoxalin-6-yl ] ethane-1, 2-diamine is described as compound 4 in WO2011/135376, and can be prepared according to the scheme described therein for compound 4.

Intermediate 2 or N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1H-pyrazol-4-yl) quinoxalin-6-yl ] ethane-1, 2-diamine is described in WO2011/135376 as the free base compound 137, and can be prepared according to the protocol described therein for compound 137.

Intermediate 3 or N- (3, 5-dimethoxyphenyl) -N' - (1-methyl) -N- [3- (1-ethyl-1H-pyrazol-4-yl) quinoxalin-6-yl ] ethane-1, 2-diamine is described as compound 449 in WO2011/135376 and can be prepared according to the scheme described therein for compound 449.

Intermediate 4 or N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1-ethyl-1H-pyrazol-4-yl) quinoxalin-6-yl ] ethane-1, 2-diamine is described in WO2011/135376 as the free base or HCl salt of compound 135, and can be prepared according to the protocol described therein for compound 135.

Intermediate 5 or N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1-methyl-1H-pyrazol-4-yl) quinoxalin-6-yl ] propane-1, 3-diamine is described as compound 382 in WO2011/135376 and can be prepared according to the scheme described therein for compound 382.

Intermediate 6 or 7-bromo-2- (1-methyl-1H-pyrazol-4-yl) -quinoxaline is described as intermediate 2 in WO2011/135376, and can be prepared according to the scheme described therein for intermediate 2.

WO2011/135376 is incorporated herein by reference.

Example A1

a) Preparation of intermediate 7

A mixture of intermediate 6 (5g; 17mmol), 2-fluoro-3, 5-dimethoxyaniline (3.6g; 21mmol), sodium tert-butoxide (5g; 52mmol) and rac-bis (diphenylphosphino) -1, 1' -dinaphthalene (0.54g; 0.87mmol) in dioxane (100mL) was degassed at room temperature under a stream of nitrogen. After 10 minutes, palladium (II) acetate (388mg; 1.7mmol) was added dropwise at room temperature under a stream of nitrogen. The reaction mixture was heated at 95 ℃ for 5 hours. The reaction mixture was cooled to room temperature and poured onto ice water and DCM. Passing the mixture through celite^®The pad is filtered. The organic layer was separated over MgSO₄Dried, filtered and evaporated to dryness. The residue was crystallized from diethyl ether and the precipitate was filtered off and dried under vacuum to give 4g (61%) of intermediate 7.

b) Preparation of intermediate 8

Sodium hydride (0.21g; 5.35mmol) was added to a solution of intermediate 7 (0.7g;1.85mmol) in DMF (25mL) at 5 ℃ under a stream of nitrogen. The mixture was stirred at 5 ℃ for 1 hour. (2-Bromoethoxy) -tert-butyldimethylsilane (0.51mL; 2.40mmol) was added dropwise at 5 ℃ under a stream of nitrogen and the reaction mixture was stirred at room temperature for 24 h. The mixture was poured into cold water and the product was extracted with EtOAc. H for organic layer₂O washing over MgSO₄Drying, filtration and evaporation gave 1.2 g (quantitative) of intermediate 8. The crude product does not need to be any pureThe reaction was used in the next step.

c) Preparation of intermediate 9

Tetrabutylammonium fluoride (1M in THF) (2mL; 2mmol) was added to a solution of intermediate 8 (1g; 1.85mmol) in THF (20mL), and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was partitioned between water and EtOAc. The organic layer was washed with brine, over MgSO₄Dried, filtered and evaporated to dryness. The residue (1.2 g) was purified by silica gel chromatography (random SiOH, 15-40 μm; 80 g; eluent: 98% DCM, 2% MeOH, 0.1% NH)₄OH). The pure fractions were collected and the solvent was evaporated. The residue (500 mg) was crystallized from diethyl ether. The precipitate was filtered and dried to yield 410 mg (52%) of intermediate 9. MP 172 deg.C (K).

d) Preparation of intermediate 10

Methanesulfonyl chloride (0.3mL; 3.88mmol) was added dropwise to a solution of intermediate 9 (547mg; 1.29mmol) and triethylamine (0.9mL; 6.46mmol) in DCM (15mL) at 5 deg.C. The reaction mixture was stirred at this temperature for 1 hour, diluted with DCM and poured to 10% K₂CO₃And (3) adding the mixture to an aqueous solution. The organic layer was decanted and MgSO₄Drying, filtering and evaporating to obtain 850mg (C:)>100%) of intermediate 10. The crude product was used without purification in the next step.

e) Preparation of intermediate 11

Intermediate 10 (0.648g; 1.29mmol) and isopropylamine (2.4mL; 28 mmol) in CH₃The mixture in CN (15mL) was heated in a sealed tube at 100 ℃ for 24 hours. The reaction mixture was cooled to room temperature, diluted with DCM, and poured onto water. The organic layer was decanted and MgSO₄Dried, filtered and evaporated to dryness. The residue was purified by chromatography on silica gel (random SiOH;24 g; gradient: from 3% MeOH, 97% DCM to 10% MeOH, 90)% DCM). The pure fractions were collected and evaporated to yield 452mg (75%) of intermediate 11.

B. Preparation of Compounds of formula (I)

Example B1:

preparation of Compound 1

Mixing N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1-methyl-1H-pyrazol-4-yl) quinoxalin-6-yl]A solution of ethane-1, 2-diamine (intermediate 1) (0.42g; 0.9mmol), formaldehyde (37% solution in water; 0.21mL;2.8mmol) in dioxane (8mL) was stirred at room temperature for 3 days. Water and EtOAc were added. The organic layer was decanted, washed with water and MgSO₄Dried and evaporated to dryness. The residue (0.52 g) was purified by chromatography on silica gel (stationary phase: 5 μm150 X30.0mm spherical bare silica, mobile phase: from 0.2% NH)₄OH, 98% DCM, 2% MeOH to 1.2% NH₄OH, 88% DCM,12% MeOH gradient). The fractions containing the desired product were collected and evaporated to dryness. The residue (0.37 g) was taken up in MeOH and Et₂The mixture of O crystallizes. The precipitate was filtered off and dried to yield 0.27 g (64%) of compound 1 (MP: 190 ℃ (DSC)).

Example B2:

preparation of Compound 2

N- (3, 5-dimethoxyphenyl) -N' - (1-methylethyl) -N- [3- (1H-pyrazol-4-yl) quinoxalin-6-yl]A solution of ethane-1, 2-diamine (intermediate 2) (0.24g; 0.52mmol), formaldehyde (37% solution in water; 0.12mL;1.55mmol) in dioxane (8mL) was stirred at room temperature for 3 days without conversion. Adding K₂CO₃(0.22g;1.55mmol) and the solution is stirred further at room temperature for 3 days. Extracting the organic layer over MgSO₄Dried and evaporated to dryness. The residue (0.176g) was purified by chromatography on silica gel (stationary phase: 5 μm150 X30.0mm spherical bare silica, mobile phase: from 0.2% NH)₄OH, 98% DCM, 2% MeOH to 1.3% NH₄OH, 87% DCM, 13% MeOH gradient). The fractions containing the desired product were collected and evaporated to dryness. The residue (79 mg) was lyophilized from acetonitrile/water 20/80 to give 66mg (34%) of compound 2 as a yellow gummy powder.

Example B3:

preparation of Compounds 3 and 4

50 μ M of intermediate 1 was incubated with 12,000g of portions from rat liver at 37 ℃ for 60 minutes at 1 mg/ml protein. A 10 mM stock solution of intermediate 1 in methanol was prepared and diluted 200-fold (0.25 ml/50ml) in incubation medium (final methanol concentration at incubation 0.5%). The incubation buffer contained 1mM EDTA, 5mM MgCl₂And 100 mM potassium phosphate buffer (pH 7.4). The reaction was started by adding NADP (1mM final concentration). The incubation was terminated by rapid freezing on dry ice.

The resulting metabolite was initially extracted with ethyl acetate. The metabolite fractions were evaporated to dryness, redissolved in DMSO: water (1:1, v/v) and separated using reversed-phase UPLC. Separation was achieved using two interchem Strategy C18-2, 2.2 μm, (150mm x 3.0 mm ID) columns using a linear gradient of solvent a over 20 minutes with 5-70% B at 0.8 ml/min. The solvent consisted of solvent A25 mM ammonium acetate pH4.0 and solvent B acetonitrile/methanol (60/40, v/v). The peak fractions corresponding to the desired product were collected and evaporated to dryness to give compounds 3 and 4.

Compound 3

Compound 3 can also be prepared according to the scheme described in example 1 starting from compound 645 of WO 2011/135376.

Compound 4

Alternatively, compounds 3 and 4 can also be prepared as follows:

boron tribromide (1M in DCM; 6 mL; 6mmol) was added dropwise to Compound 1(485 mg; 1.06mmol) in DCM (25M) at 5 ℃ under a stream of nitrogenL) solution. The solution was slowly warmed to room temperature and stirred for 1.5 hours. The reaction mixture was diluted with DCM, poured onto brine, and taken up with solid K₂CO₃Alkalizing. The organic layer was separated, washed with brine, over MgSO₄Dried, filtered and evaporated to dryness. The residue was purified by chromatography on silica gel (random SiOH, 40g; mobile phase: from 0.5% NH)₄OH, 94.5% DCM, 5% MeOH to 0.5% NH₄OH, 89.5% DCM, 10% MeOH gradient). The fractions containing the product were collected and evaporated to dryness to give 110 mg (23%) of compound 1 and 287 mg of a mixture of compounds 3 and 4. This latter fraction was purified by achiral SFC (Chiralpak AD-H5 μm 250 x30 mn; mobile phase: 0.3% isopropylamine, 70% CO₂30% MeOH). The pure fractions were collected, concentrated and recovered from Et₂And (4) crystallizing the O/CAN. The precipitate was filtered to give 53 mg (11%) of Compound 3(MP: 255 ℃, K) and 148 mg (31%) of Compound 4 (MP: 256 ℃, K).

Example B4:

a 2mM stock solution of intermediate 1 was prepared in methanol and diluted 200-fold in incubation medium (final methanol concentration 0.5% at incubation). Incubate 12,000g of a fraction from rat liver at 37 ℃ with 1 mg/ml protein for 60 minutes. The incubation buffer contained 1mM EDTA, 5mM MgCl₂And 100 mM potassium phosphate buffer (pH 7.4). The reaction was started by adding NADP (1mM final concentration). The incubation was terminated by rapid freezing on dry ice.

The resulting incubation (1 ml) was mixed with 5 volumes of acetonitrile, vortexed and sonicated for 10 minutes. Proteins were removed by centrifugation at 3200 rpm for 30 minutes at 8 ℃. The supernatant was removed and evaporated to dryness at 30 ℃ under a stream of nitrogen. The extract was redissolved in acetonitrile/water (1:1, v/v). The samples were analyzed as follows:

UPLC and MS detection

-ultra-performance liquid chromatography pump acquisition Binary Solvent Manager/Waters 2777 CTC-Pal-syringe

-a UV detector: waters Acquity PDA

-an MS detector: waters G2(S) QToF MS/Thermo LTQ-Orbitrap

-a data system: waters Masslynx 4.1

The operating conditions are as follows:

-a column: interchim, Strategy C18-2, 2.2 μm 2x (150mm x 3.0 mm ID)

Column temperature: t = 60 deg.C

-sample temperature: t = 10 deg.C

-a mobile phase: solvent A: 0.025M ammonium acetate pH4.0 solvent B: 60/40 (v/v) acetonitrile/methanol

-elution mode:

linear gradient:

-run time: 30 min

-flow rate: 0.8 ml/min

Injecting by using an injector:

-a mobile phase: acetonitrile water (1:1, v/v)

-flow rate: 5 mu l/min

Detection conditions

MS Condition-waters synapt g2 and g2s Mass Spectroscopy

MS analysis was performed using Waters SYNAPT G2 and G2S mass spectrometers equipped with binary electrospray ionization probes, and operated in high resolution, positive ion mode. The capillary voltage was set to 3 kV and the cone voltage to 40V. The source temperature was 120 ℃ and the desolvation temperature was 400 ℃. The mass spectrometer was calibrated with a sodium formate solution delivered by a sample nebulizer. LockSpray^TMThe ESI probe provided an independent source of the lock mass calibrator leucine enkephalin. In thatm/z556.2771 acts as a lock mass in the all-MS and MSMS modes. QTOF data (MS, MSMS) were obtained in centroid mode with variable scan time (0.5-1.0 sec). All data were processed using Masslynx software.

MS condition-thermal ltq-orbitrap mass spectrometer

LTQ-Orbitrap mass spectrometers are equipped with an electrospray ionization source operating in positive ion mode. Accurate mass measurement using external calibration or lock mass calibration ((ii))m/z391.2843 lock mass ions). For maximum sensitivity, 10 ng/mu L of unchanged drug standard solution is used for adjustmentAnd (4) saving source parameters. Same solution used for limiting to MSⁿOptimal collision energy used during fragmentation. For MSⁿFragmentation metabolites were selected from LC-MS traces using data-dependent scanning. Data is acquired in centroid mode and processed using XCalibur software.

Compound 4 ([ MH ] + m/z 445), Compound 3 ([ MH ] + m/z 445) and Compound 5 ([ MH ] + m/z 431) were detected in the above experiment.

Compound 5:

example B5

Preparation of Compound 6

A solution of intermediate 3 (292 mg; 0.675 mmol), formaldehyde (37% aqueous solution; 151. mu.L; 2.02 mmol) in 1, 4-dioxane (5.48 mL) was stirred at room temperature for 3 days. As low conversion was noted, additional formaldehyde (37% aqueous solution; 252. mu.L; 3.37mmol) was added and the reaction mixture was stirred at 70 ℃ for 16 hours. Addition of H₂O and EtOAc. The organic layer was decanted and MgSO₄Dried, filtered and evaporated to dryness.

The residue (0.325g) was purified by chromatography on silica gel (random SiOH, 40g, mobile phase: from 95% DCM, 5% MeOH, 0.5% NH)₄OH to 90% DCM, 10% MeOH, 1% NH₄OH gradient). Fractions containing product were mixed and concentrated to give an intermediate fraction (106 mg) from Et₂Crystallization of a mixture of O/CAN, filtration and drying gave 86 mg (28%) of Compound 6. MP 170 deg.C (K).

Example B6

As the HCl salt (1.65HCl 2.2H)₂O) preparation of Compound 7

Intermediate 4 (293 mg; 0638 mmol), formaldehyde (37% in water)Solution 143 μ L, 1.91mmol) in 1, 4-dioxane (5.16 mL) was stirred at room temperature for 3 days. Since no conversion was noted, additional formaldehyde (37% aqueous solution; 238. mu.L; 3.18 mmol) was added and the reaction mixture was stirred at 70 ℃ for 16 hours. Additional formaldehyde (37% aqueous solution; 477. mu.L; 6.36mmol) was again added and the reaction mixture was stirred at 70 ℃ for 16 hours. Addition of H₂O and EtOAc. The organic layer was decanted and MgSO₄Dried, filtered and evaporated to dryness.

The residue (0.48g) was purified by chromatography on silica gel (5 μm150 X30.0mm spherical bare silica, mobile phase: from 0.2% NH₄OH, 98% DCM, 2% MeOH to 1% NH₄OH, 90% DCM, 10% MeOH gradient). Fractions containing the product were mixed and concentrated to give 148 mg of intermediate fraction which was purified by achiral SFC (stationary phase: CYANO 6 μm150x21.2mm, mobile phase: 90% CO)₂, 10% MeOH(0.3% iPrNH₂)). Fractions containing product were mixed and concentrated to give 100mg of intermediate fraction, which was dissolved in MeOH. 0.1mL of HCl/iPrOH (2-5N) was added at 0 ℃. The mixture was then concentrated and the residue obtained was dissolved in Et₂And O. The precipitate was filtered and dried to give 103 mg (29%) of compound 7 as a red solid. MP 152 deg.C (K).

Example B7

Preparation of Compound 8

A solution of intermediate 11 (382mg; 0.82mmol) and formaldehyde (37% in water; 308 μ L; 4.11mmol) in dioxane (10mL) was heated at 60 ℃ for 3 days. Addition of H₂O and EtOAc. The organic layer was decanted and MgSO₄Dried, filtered and evaporated to dryness. The residue was purified by chromatography on silica gel (spherical bare silica 5 μm150X 30.0 mm; gradient: from 71% heptane, 1% MeOH (+10% NH)₄OH), 28% EtOAc to 0% heptane, 20% MeOH (+10% NH)₄OH), 80% EtOAc). The pure fractions were collected and evaporated to dryness. The residue (65 mg) was purified by reverse phase chromatography (X-Bridge-C185 μm 30X 150 mm; gradient: from 80% NH)₄HCO₃0.5%, 20% CH₃CN to 0% NH₄HCO₃0.5%, 100% CH₃CN). The pure fractions were collected and evaporated to yield 15 mg (4%) of compound 8. And (5) MP of 266 ℃ (K).

Example B8

Preparation of Compound 9

A solution of intermediate 5 (0.21g; 0.46mmol) and formaldehyde (37% in water; 0.1mL; 1.4mmol) in 1, 4-dioxane (8mL) was stirred at room temperature for 3 days. After one week, additional formaldehyde (37% aqueous solution; 0.5mL;20.55mmol) was added and the mixture was stirred at room temperature for an additional 2 days. Addition of H₂O and EtOAc. Extracting the organic layer over MgSO₄Dried and evaporated to dryness.

The resulting residue (170 mg) was purified by reverse phase chromatography (stationary phase: X-Bridge-C185 μm 30X 150mm, mobile phase: from 85% NH)₄HCO₃0.5%, 15% CAN to 0% NH₄HCO₃0.5%, 100% CAN gradient). Fractions containing the product were combined and concentrated to give an intermediate fraction (10 mg) which was lyophilized with acetonitrile/water 20/80 to give 9mg (4%) of compound 9 as a yellow powder. MP is gum at 80 deg.C (K).

Analysis section

LCMS (liquid chromatography/mass spectrometry) (see Table A1)

LC measurements were performed using a UPLC (ultra performance liquid chromatography) acquity (waters) system comprising a binary pump with degasser, an autosampler, a Diode Array Detector (DAD) and a column as indicated in the respective methods below, which was maintained at a temperature of 40 ℃. The flow from the column was carried to the MS detector. The MS detector was configured with an electrospray ionization source. On a Quattro (triple quadrupole mass spectrometer from Waters), the capillary tip voltage was 3 kV and the source temperature was maintained at 130 ℃. Nitrogen was used as the nebulizer gas. Data acquisition was performed using a Waters-Micromass MassLynx-Openlynx data System.

The reverse phase UPLC was performed on a Waters Acquity BEH (bridged ethylsiloxane/silica hybrid) C18 column (1.7 μm, 2.1 x 100 mm) at a flow rate of 0.343 ml/min. Two mobile phases (mobile phase A: 95% 7mM ammonium acetate/5% acetonitrile; mobile phase B: 100% acetonitrile) were used to run the following gradient conditions: from 84.2% a and 15.8% B (held for 0.49 min) over 2.18 min to 10.5% a and 89.5% B, held for 1.94 min and returned to the starting condition over 0.73 min, held for 0.73 min. Injection volume 2 μ l was used. The cone voltage was 20V for positive and negative ionization modes. Mass spectra were acquired from 100 to 1000 scans using an intermediate scan delay of 0.1 seconds over 0.2 seconds.

DSC

For many compounds, Melting Point (MP) was determined by DSC1 (Mettler-Toledo). Melting points were measured with a temperature gradient of 10 ℃ per minute. The maximum temperature is 350 ℃. The value is the peak value.

For many compounds, melting points were obtained with a Kofler hot plate consisting of a hot plate with a linear temperature gradient, a sliding pointer, and a temperature scale in degrees celsius.

NMR

For compounds 1,2, 6-9, NMR experiments were performed using Bruker Avance III 500 using internal deuterium locks and equipped with reverse triple resonance: (¹H,¹³C,¹⁵N TXI) probe head. Chemical shifts () are reported in parts per million (ppm).

For compounds 3 and 4, each fraction was dissolved in 250 μ l of anhydrous DMSO-d6 and the resulting solution was transferred to a 5mm Shigemi NMR tube matched to the respective solvent with magnetic sensitivity.

The experiments were recorded on a Bruker Avance 600 MHz spectrometer equipped with a reverse detection 5-mm cryoprobe (CPTCI). Recording 1D¹H and 2D NOESY, HSQC and HMBC spectra, a standard Bruker pulse program was run. NOESY spectroscopy was used to determine total-space connectivity (through-space connectivity); HMBC spectroscopy is used to detect all-bond connectivity(s). Chemical shifts () are reported in ppm. Using the center of the DMSO-D5 multiplet at 2.50 ppm or the center of the acetonitrile-D2 multiplet at 1.94 ppm as internal reference, from 1D¹H spectral acquisition¹HNMR chemical shift data. The coupling constant is measured in Hz.¹³C NMR chemical shifts used at 39.51 ppmThe center of the DMSO-d6 multiplet was obtained as an internal reference.

Table a 1:co. No. means the compound number; retention time (R)_t) Per minute; MP means melting point (. degree.C.).

As understood by those skilled in the art, the compounds synthesized using the indicated schemes may exist as solvates, e.g., hydrates, and/or contain residual solvent or fewer impurities.

Compound 1

¹H NMR at 350 ℃ K

¹H NMR (500 MHz, DMSO-d ₆) ppm 0.99 (d,J=6.5 Hz, 6 H) 2.84 (spt,J=6.5 Hz, 1 H) 2.88 - 2.93 (m, 2 H) 3.56 (br. s., 2 H) 3.76 (s, 3 H) 3.82 -3.91 (m, 5 H) 3.93 (s, 3 H) 6.42 (d,J=2.2 Hz, 1 H) 6.57 (d,J=2.2 Hz, 1 H)6.97 (d,J=2.7 Hz, 1 H) 7.26 (dd,J=9.1, 2.7 Hz, 1 H) 7.75 (d,J=9.1 Hz, 1 H)8.14 (s, 1 H) 8.46 (s, 1 H) 8.87 (s, 1 H)

Compound 2

¹H NMR at 350 ℃ K

¹H NMR (500 MHz, DMSO-d ₆) ppm 0.99 (d,J=6.6 Hz, 6 H) 2.84 (spt,J=6.6 Hz, 1 H) 2.88 - 2.95 (m, 2 H) 3.56 (br. s., 2 H) 3.76 (s, 3 H) 3.80 -3.95 (m, 5 H) 6.43 (d,J=2.2 Hz, 1 H) 6.57 (d,J=2.2 Hz, 1 H) 6.99 (d,J=2.7Hz, 1 H) 7.26 (dd,J=9.5, 2.7 Hz, 1 H) 7.75 (d,J=9.5 Hz, 1 H) 8.35 (br. s.,2 H) 8.92 (s, 1 H) 13.08 (br. s., 1 H)

Compound 3

¹H NMR at 300 ℃ K

¹H NMR (600 MHz, DMSO-d ₆) ppm 0.98 (d,J=6.42 Hz, 6 H) 2.82 (spt,J=6.50 Hz, 1 H) 2.88 (t,J=4.53 Hz, 2 H) 3.69 (s, 3 H) 3.91 (s, 3 H) 6.29 (d,J=2.27 Hz, 1 H) 6.42 (d,J=2.27 Hz, 1 H) 6.89 (br. s., 1 H) 7.25 (br. s., 1 H)7.75 (d,J=9.07 Hz, 1 H) 8.18 (s, 1 H) 8.53 (s, 1 H) 8.89 (s, 1 H)

Compound 4

¹H NMR at 300 ℃ K

¹H NMR (600 MHz, DMSO-d ₆) ppm 0.96 (d,J=6.70 Hz, 6 H) 2.81 (spt,J=6.70 Hz, 1 H) 2.86 (t,J=4.34 Hz, 2 H) 3.79 (s, 3 H) 3.91 (s, 3 H) 6.26 (d,J=1.89 Hz, 1 H) 6.41 (d,J=2.27 Hz, 1 H) 6.91 (br. s., 1 H) 7.27 (br. s., 1 H)7.75 (d,J=9.44 Hz, 1 H) 8.18 (s, 1 H) 8.53 (s, 1 H) 8.89 (s, 1 H)

Compound 6

¹H NMR at 300 ℃ K

¹H NMR (500 MHz, DMSO-d ₆) ppm 1.43 (t,J=7.3 Hz, 3 H) 2.22 (br s, 3H) 2.82 (br s, 2 H) 3.50 - 4.10 (m, 10 H) 4.20 (q,J=7.3 Hz, 2 H) 6.46 (d,J=1.9 Hz, 1 H) 6.59 (d,J=1.9 Hz, 1 H) 6.92 (br s, 1 H) 7.25 (br d,J=7.3 Hz, 1H) 7.77 (d,J=9.1 Hz, 1 H) 8.20 (s, 1 H) 8.58 (s, 1 H) 8.92 (s, 1 H)

Compound 7

¹H NMR at 300 ℃ K

¹H NMR (500 MHz, DMSO-d ₆) ppm 1.32 (dd,J=9.8, 6.6 Hz, 6 H) 1.44 (t,J=7.4 Hz, 3 H) 3.35 - 3.60 (m, 3 H) 3.93 (s, 3 H) 3.79 (s, 3 H) 4.22 (q,J=7.5 Hz, 2 H) 4.43 (br d,J=12.9 Hz, 1 H) 4.58 (br s, 1 H) 6.56 (d,J=2.5 Hz,1 H) 6.70 (d,J=2.2 Hz, 1 H) 7.17 (br d,J=1.9 Hz, 1 H) 7.30 (dd,J=9.3, 2.4Hz, 1 H) 7.84 (d,J=9.1 Hz, 1 H) 8.23 (s, 1 H) 8.62 (s, 1 H) 9.02 (s, 1 H)10.26 (br s, 1 H)

Compound 8

¹H NMR was performed at 350 ℃ K

¹H NMR (500 MHz, DMSO-d ₆) ppm 0.99 (d,J=6.6 Hz, 6 H) 2.85 (spt,J=6.5 Hz, 1 H) 2.92 (t,J=4.6 Hz, 2 H) 3.58 (br s, 2 H) 3.80 - 4.05 (m, 11 H)6.84 (d,J=6.9 Hz, 1 H) 6.92 (br s, 1 H) 7.20 (br d,J=9.5 Hz, 1 H) 7.79 (d,J=9.1 Hz, 1 H) 8.15 (s, 1 H) 8.46 (s, 1 H) 8.89 (s, 1 H)

Compound 9

¹H NMR at 300 ℃ K

¹H NMR (500 MHz, DMSO-d ₆) ppm 0.97 (br d,J=5.4 Hz, 6 H) 1.64 (br s,2 H) 2.72 (br s, 2 H) 2.84 (spt,J=6.4 Hz, 1 H) 3.50 - 3.80 (m, 7 H) 3.84 (s,3 H) 3.92 (s, 3 H) 6.21 (d,J=2.2 Hz, 1 H) 6.61 (d,J=2.5 Hz, 1 H) 6.92 (brs, 2 H) 7.71 (d,J=9.5 Hz, 1 H) 8.19 (s, 1 H) 8.54 (s, 1 H) 8.91 (s, 1 H)

Diaza derivatives

Some of the signal of the loop was broadened beyond the detection of the spectrum measured at 300K in DMSO-d 6.

Pharmacological moieties

Bioassay method A

FGFR1 (enzyme assay)

FGFR1 (h) (25ng/ml) was treated with 50mM HEPES pH 7.5, 6mM MnCl in the presence of compound (1% DMSO final) in a final reaction volume of 30. mu.L₂、1 mM DTT、0.1 mM Na₃VO₄0.01% Triton-X-100, 500 nM Btn-Flt3 and 5 μ M ATP incubations. After incubation at room temperature for 60 minutes, the reaction was stopped with 2.27 nM EU-anti-P-Tyr, 7mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA left at room temperature for 60 minutes. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals (ex340 nm. Em 620 nm, em 655 nm) were then measured and the results expressed in RFU (relative fluorescence units). In this assay, inhibition of different compound concentrations (range 10 μ M to 0.1 nM) was determined and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

FGFR2 (enzyme assay)

FGFR2 (h) (150ng/ml) was treated with 50mM HEPES pH 7.5, 6mM MnCl in the presence of compound (1% DMSO final) in a final reaction volume of 30. mu.L₂、1 mM DTT、0.1 mM Na₃VO₄0.01% Triton-X-100, 500 nM Btn-Flt3 and 0.4 μ M ATP incubation. After incubation at room temperature for 60 minutes, the reaction was stopped with 2.27 nM EU-anti-P-Tyr, 7mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA left at room temperature for 60 minutes. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals (ex340 nm. Em 620 nm, em 655 nm) were then measured and the results expressed in (relative fluorescence units). In this assay, inhibition was measured at different compound concentrations (range 10 μ M to 0.1 nM) and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

FGFR3 (enzyme assay)

FGFR3 (h) (40ng/ml) was treated with 50mM HEPES pH 7.5, 6mM MnCl in the presence of compound (1% DMSO final) in a final reaction volume of 30. mu.L₂、1 mM DTT、0.1 mM Na₃VO₄0.01% Triton-X-100, 500 nM Btn-Flt3 and 25 μ M ATP incubations. After incubation at room temperature for 60 minutes, the reaction was stopped with 2.27 nM EU-anti-P-Tyr, 7mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA left at room temperature for 60 minutes. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals (ex340 nm. Em 620 nm, em 655 nm) were then measured and the results expressed in RFU (relative fluorescence units). In this assay, inhibition was measured at different compound concentrations (range 10 μ M to 0.1 nM) and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

FGFR4 (enzyme assay)

FGFR4 (h) (60ng/ml) was treated with 50mM HEPES pH 7.5, 6mM MnCl in the presence of compound (1% DMSO final) in a final reaction volume of 30. mu.L₂、1 mM DTT、0.1 mM Na₃VO₄0.01% Triton-X-100, 500 nM Btn-Flt3 and 5 μ M ATP incubations. After incubation at room temperature for 60 minutes, 2.27 nM at room temperature for 60 minutesEU-anti-P-Tyr, 7mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA stop the reaction. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals (ex340 nm. Em 620 nm, em 655 nm) were then measured and the results expressed in RFU (relative fluorescence units). In this assay, inhibition was measured at different compound concentrations (range 10 μ M to 0.1 nM) and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

KDR (VEGFR2) (enzyme assay)

KDR (h) (150ng/ml) was treated with 50mM HEPES pH 7.5, 6mM MnCl in the presence of compound (1% DMSO final) in a final reaction volume of 30. mu.L₂、1 mM DTT、0.1 mM Na₃VO₄0.01% Triton-X-100, 500 nM Btn-Flt3 and 3 μ M ATP incubations. After incubation at room temperature for 120 minutes, the reaction was stopped with 2.27 nM EU-anti-P-Tyr, 7mM EDTA, 31.25 nM SA-XL-665 and 0.02% BSA left at room temperature for 60 minutes. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals (ex340 nm. Em 620 nm, em 655 nm) were then measured and the results expressed in RFU (relative fluorescence units). In this assay, inhibition was measured at different compound concentrations (range 10 μ M to 0.1 nM) and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

Ba/F3-FGFR1 (without IL3 or with IL3) (cell proliferation assay)

100 nl of compound diluted in DMSO was sprayed in 384 well plates before adding 50 μ L of cell culture medium (phenol red free RPMI-1640, 10% FBS, 2mM L-glutamine and 50 μ g/ml gentamicin) containing 20000 cells/well of Ba/F3-FGFR1 transfected cells. Place the cells in a chamber at 37 ℃ and 5% CO₂The incubator below. After 24 hours, 10. mu.l of Alamar Blue solution (0.5 mM K)₃Fe(CN)₆、0.5 mM K₄Fe(CN)₆0.15 mM resazurin and 100 mM phosphate buffer) were added to each well at 37 ℃ and 5% CO₂After 4 hours of incubation, RFU (relative fluorescence units) was measured in a fluorescence plate reader (ex. 540 nm., em. 590 nm.).

In this assay, the inhibitory effect of different compound concentrations (range 10. mu.M to 0.1 nM) was determined andfor computing IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

As a counter screen (counterscreen), the same experiment was performed in the presence of 10 ng/ml murine IL 3.

Ba/F3-FGFR3 (without IL3 or with IL3) (cell proliferation assay)

100 nl of compound diluted in DMSO was sprayed in 384 well plates before adding 50 μ L of cell culture medium (phenol red free RPMI-1640, 10% FBS, 2mM L-glutamine and 50 μ g/ml gentamicin) containing 20000 cells/well of Ba/F3-FGFR3 transfected cells. Place the cells in a chamber at 37 ℃ and 5% CO₂The incubator below. After 24 hours, 10. mu.l of Alamar Blue solution (0.5 mM K)₃Fe(CN)₆、0.5 mM K₄Fe(CN)₆0.15 mM resazurin and 100 mM phosphate buffer) were added to each well at 37 ℃ and 5% CO₂After 4 hours of incubation, RFU (relative fluorescence units) was measured in a fluorescence plate reader (ex. 540 nm., em. 590 nm.).

In this assay, inhibition of different compound concentrations (range 10 μ M to 0.1 nM) was determined and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

As a counter-screen, the same experiment was performed in the presence of 10 ng/ml murine IL 3.

Ba/F3-KDR (No IL3 or with IL3) (cell proliferation assay)

100 nl of compound diluted in DMSO was sprayed in 384 well plates before adding 50 μ L of cell culture medium (phenol red free RPMI-1640, 10% FBS, 2mM L-glutamine and 50 μ g/ml gentamicin) containing 20000 cells/well of Ba/F3-KDR transfected cells. Place the cells in a chamber at 37 ℃ and 5% CO₂The incubator below. After 24 hours, 10 μ l of Alamar Blue solution (0.5 mM K)₃Fe(CN)₆、0.5 mM K₄Fe(CN)₆0.15 mM resazurin and 100 mM phosphate buffer) were added to each well at 37 ℃ and 5% CO₂After 4 hours of incubation, RFU (relative fluorescence units) was measured in a fluorescence plate reader (ex. 540 nm., em. 590 nm.).

In this assay, the concentration of different compounds is determined(range 10. mu.M to 0.1 nM) and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

As a counter-screen, the same experiment was performed in the presence of 10 ng/ml murine IL 3.

Ba/F3-FGFR4 (cell proliferation assay)

100 nl of compound diluted in DMSO was sprayed in 384 well plates before adding 50 μ L of cell culture medium (phenol red free RPMI-1640, 10% FBS, 2mM L-glutamine and 50 μ g/ml gentamicin) containing 20000 cells/well of Ba/F3-FGFR4 transfected cells. Place the cells in a chamber at 37 ℃ and 5% CO₂The incubator below. After 24 hours, 10. mu.l of Alamar Blue solution (0.5 mM K)₃Fe(CN)₆、0.5 mM K₄Fe(CN)₆0.15 mM resazurin and 100 mM phosphate buffer) were added to each well at 37 ℃ and 5% CO₂After 4 hours of incubation, RFU (relative fluorescence units) was measured in a fluorescence plate reader (ex. 540 nm., em. 590 nm.).

In this assay, inhibition of different compound concentrations (range 10 μ M to 0.1 nM) was determined and used to calculate IC₅₀(M) and pIC₅₀(-logIC₅₀) The value is obtained.

Data for the compounds of the invention in the above assays are provided in table a 2.

TABLE A2

Biological assay B

^®Enzyme binding assay (KINOMEscan)

Kinase enzyme binding affinity of Compounds disclosed herein Using KINOMEscan^®Technical determination, which was done by DiscoveRx Corporation, San Diego, California, USA (www.kinomescan.com). Table a3 reports the Kd values (nM) obtained, where Kd is the inhibitor binding constant:

TABLE A3

Claims (40)

1. A compound of formula (I):

(I)

including any tautomeric or stereochemically isomeric form thereof, wherein

n represents an integer equal to 1 or 2;

R₁represents hydrogen, C_1-6Alkyl, hydroxy C_1-6Alkyl, with-C (= O) NHCH₃Substituted C_1-6Alkyl or by-S (= O)₂- C_1-4Alkyl substituted C_1-6An alkyl group;

R_2arepresents hydrogen, fluorine or chlorine;

R_2bor R_2cEach independently represents a methoxy group or a hydroxyl group;

R₃represents hydrogen, C_1-6Alkyl radical, C_3-6Cycloalkyl radicals or with C_3-6Cycloalkyl-substituted C_1-2An alkyl group;

R₄represents hydrogen, methyl or ethyl;

or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound has the structure

(Ia)。

3. The compound of claim 1 or 2, wherein R_2aRepresents hydrogen or fluorine.

4. The compound of claim 3, wherein R_2aRepresents fluorine.

5. The compound of claim 1, wherein the compound has the structure

(Ib)。

6. A compound according to claim 1, wherein n represents an integer equal to 1.

7. The compound of claim 1, wherein R₃Represents hydrogen.

8. The compound of claim 1, wherein R₃Is represented by C_1-6An alkyl group.

9. The compound of claim 8, wherein R₃Is represented by C_1-4An alkyl group.

10. The compound of claim 1, wherein the compound has the structure

(Ic)。

11. The compound of claim 1, wherein R₁Represents hydrogen or C_1-6An alkyl group.

12. The compound of claim 11, wherein R₁Is represented by C_1-6An alkyl group.

13. The compound of claim 12, wherein R₁Represents a methyl group.

14. The compound of claim 1, wherein R_2bRepresents a methoxy group.

15. The compound of claim 1, wherein R_2bRepresents a hydroxyl group.

16. The compound of claim 1, wherein R_2cRepresents a methoxy group.

17. The compound of claim 1, wherein R_2cRepresents a hydroxyl group.

18. The compound of claim 1, wherein said compound is

Or a pharmaceutically acceptable salt thereof.

19. The compound of claim 18, wherein said compound is

。

20. The compound of claim 1, wherein said compound is selected from the group consisting of

；

(ii) a And

。

21. a process for the preparation of a compound of formula (I) according to claim 1, which process comprises:

reacting a compound of formula (II)

With formaldehyde in the presence of a suitable solvent at a suitable temperature,

wherein R is₁、R_2a、R_2b、R_2c、R₃、R₄And n is as defined in claim 1.

22. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 20.

23. Use of a compound as defined in any one of claims 1 to 20 for the manufacture of a medicament for the prevention or treatment of a disease state or condition mediated by a FGFR kinase.

24. Use of a compound as defined in any one of claims 1 to 20 for the manufacture of a medicament for the prevention or treatment of cancer.

25. The use of claim 24, wherein the cancer is bladder cancer, urothelial cancer, breast cancer, glioblastoma, lung cancer, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, head and neck squamous cell carcinoma, gastric cancer, esophageal cancer, cholangiocarcinoma, hepatocellular carcinoma.

26. The use of claim 25, wherein the cancer is esophageal cancer.

27. The use of claim 26, wherein the cancer is esophageal squamous cell carcinoma, adenocarcinoma of the esophagus.

28. The use of claim 25, wherein the cancer is urothelial cancer.

29. The use of claim 28, wherein the cancer is metastatic urothelial cancer or urothelial cancer that cannot be resected following surgery.

30. The use of claim 25, wherein the cancer is lung cancer.

31. The use of claim 30, wherein the cancer is non-small cell lung cancer, squamous cell lung cancer, small cell lung cancer, adenocarcinoma of the lung.

32. The use of claim 25, wherein the cancer is bladder cancer.

33. The use of claim 32, wherein the cancer is bladder cancer with a FGFR3 chromosomal translocation.

34. The use of claim 32, wherein the cancer is bladder cancer having a FGFR3 point mutation.

35. The use of claim 24, wherein

(i) The cancer is a tumor having FGFR1, FGFR2, FGFR3, or a mutant of FGFR 4; or (ii) the cancer is a tumor having an gain-of-function mutant of FGFR2 or FGFR 3; or (iii) the cancer is a tumor with overexpression of FGFR 1.

36. The use of claim 25, wherein the cancer is cholangiocarcinoma.

37. The use of claim 24, wherein the compound is

。

38. The use of any one of claims 25-36, wherein the compound is

。

39. Use of a compound as defined in any one of claims 1 to 20 in the manufacture of a medicament for inhibiting an FGFR kinase.

40. Use of a compound as defined in claim 19 in the manufacture of a medicament for inhibiting an FGFR kinase.